Barley transformation experiment by gene gun method
Microparticle bombardment, also known as the gene gun method, has been widely used in the genetic transformation of cereal crops. This method brings DNA directly into plant cells by accelerating the bombardment of gold dust particles encapsulating plasmid DNA. The first fully fertile transgenic barley was obtained by the gene gun method. This experiment is based on "A Guide to Transgenic Technology and Field Identification of Wheat Crops" (H.D. Jones, P.R. Hewley, ed.). H.D. Jones P.R. Hewlett, eds.
Operation method
Gene Shot Transformation Barley
Materials and Instruments
Eggs of wheat Move 1. Preparation of tissue culture medium For more product details, please visit Aladdin Scientific website.
Vegetable gel Ethanol Sodium hypochlorite Distilled water Spermidine
EP tube Pipette gun
( 1 ) First, prepare plant gel at 2 times the desired concentration and autoclave it (see Note 2).
( 2 ) All tissue culture media should be filter-sterilized, therefore, except for the sterile stock solution, mix the other media components at 2 times the desired concentration, adjust the pH to the appropriate level, and then filter-sterilize the media (using a bottle-top filter and 0.22 μm microporous filter membrane).
( 3 ) Heat the 2x gel and 2x medium in a water bath to 60°C before use.
( 4 ) Add the required stock solution to the hot medium under aseptic conditions, mix the medium and plant gel and pour into a 9 cm Petri dish (Sterilin).
( 5 ) For differentiation culture, use a tall petri dish (100 mmx 20 mm, Falcon).
( 6 ) For infiltration treatment, use a small 5 cm Petri dish (Sterilin).
2. Isolation of young embryos
(1) Harvesting and sterilizing the spikelets
① Harvest the ears when the diameter of the young embryos reaches 1~2 mm (see Note 3).
② Before harvesting the ears, take a seed from the middle of each ear and check the size of the young embryo to make sure that it meets the required requirements [see Chapter 9, "Agrobacterium-mediated transformation of barley", Fig. 9.2 (a ) ~ (c )].
(iii) Remove the seed from the spike and de-awning it, but do not destroy the seed husk.
④ Rinse with 70% ethanol for 30 s, then rinse with distilled water for 3 times, then soak in a solution of sodium hypochlorite (Fluka 71696) in a ratio of 50:50 with water for 4 min, then rinse with distilled water for 4 times, and finally pour off the water but keep the seeds moist, and place them in sterile screw-top bottles for use.
(2) Isolation of young embryos and removal of embryonic axes
All the operations were carried out in an ultra-clean table:
① Take about 20 sterile seeds at a time and place them on a sterile blue or black backing plate of a dissecting microscope. Hold the seeds in place with one forceps and remove the embryo axis by peeling out the young embryos with the other deterrent (see Note 4).
( 2 ) Put 25~30 young embryos on each dish of healing induction medium, with the shield facing up, and incubate in dark at 25℃.
3. Preparation of DNA-encapsulated gold dust particles
(1) Preparation of gold powder mother liquor
The method of DNA-encapsulated gold particles is similar to that in the literature [ 9 ].
Weigh 40 mg of gold powder in a 1.5 ml EP tube and add 1 ml of 100% ethanol.
② Vortex and shake to mix the gold powder well, and then centrifuge to precipitate it.
③ Pour off the ethanol and repeat step 2 twice.
④ After the last centrifugation, remove the ethanol and resuspend it by vortexing with 1 ml of sterile water.
⑤ Dispense 50 μl of gold powder into sterile EP tubes, taking care to keep the gold powder particles in suspension by constant mixing and storing at -20°C after dispensing.
(2) Preparation of spermidine
① Melt a bottle containing 1 g of spermidine (Sigma-Aldrich) in a 65°C water bath.
② Pipette 14 μl of the solution into a sterile 1.5 ml centrifuge tube and store at -20°C until ready for use.
(3) Encapsulation of gold dust particles
① Remove spermidine from the refrigerator and thaw it.
② Add 986 μl of sterile water and mix by vortexing and shaking.
③ Filter the solution into a sterile 1.5 ml centrifuge tube using a 1 ml filter and a small filter tip (Minisart 0.2 μm filter, Sartorius) to make a 0.1 mol/L spermidine solution.
④ Remove a 50 μl tube of gold powder from the refrigerator and allow it to thaw.
⑤ Add 5 μl of DNA (1 μg/μl) along the wall of the centrifuge tube containing the gold powder, and immediately vortex and shake to mix the gold powder and DNA well (see Note 5).
(6) Then add 50 μl of 2.5 mol/L CaCl2 and 20 μl of 0.1 mol/L spermidine to the cap of the centrifuge tube, close the cap, and invert the tube to mix the CaCl2 and spermidine with the gold powder pellet (do not allow the CaCl2 and spermidine to mix with the gold powder pellet before inverting the centrifuge tube), and then immediately vortex and shake the tube so that all components are well mixed.
(vii) The tubes were placed on ice for 1 min and then centrifuged at 1957 g for no more than 30 s to allow the gold powder to precipitate.
(8) Remove the supernatant, add 250 μl of 100% ethanol, and mix well by blowing gently with the tip of a gun, and then vortex and oscillate, which is a process to wash the gold dust particles embedded with DNA.
⑨ 1957 g centrifugation not more than 30 s precipitate gold powder particles, remove the supernatant, add 60 μl 100% ethanol.
The DNA-embedded gold dust particles, i.e., the microcarriers, were prepared by vortexing and resuspending.
4. Particle bombardment of young embryos
(1) Infiltration of young embryos
Before bombardment, take the young embryos after 1 day of isolation and treat them in hypertonic medium for 4 h. 20~30 young embryos per dish are placed in the center of the dish within 1.6 cm2 with the shield facing up. The embryos can be placed close together, but not touching each other. After bombardment, the young embryos were continued to be treated in hypertonic medium for 16 h.
(2) Gene gun preparation
① All components of the gene gun and the internal body should be cleaned and sterilized with 100% ethanol. The termination screen and carrier membrane should be sterilized by soaking in ethanol and then air-dried. The rupture disk is soaked in isopropyl alcohol before use and does not need to be sterilized again.
② After the carrier membrane is air-dried, put it into the carrier membrane holder first, and then aspirate 3.5 μl of DNA-encapsulated gold dust particles to the center of each carrier membrane. Be careful to keep the gold particles in suspension when pipetting.
(3) Gene Gun Parameters
The position of each component of the gene gun in the bombardment chamber is as follows:
Distance between rupture disk and carrier membrane: 2.2 cm
Distance between the carrier membrane and the termination screen: 1.3 cm
Distance between termination screen and sample holder: 5.8 cm (third stop up from the base of the gun)
Vacuum: 28 in.Hg.
(4) Particle bombardment
① Before bombarding the young embryos, the gene gun is bombarded twice in vacuo (see Note 6).
② The 1100 psi rupture disk is soaked in isopropyl alcohol and placed inside the rupture disk retaining cap, which is then attached to the nitrogen gas accelerator tube in the gene gun chamber and screwed tight at the base.
③ Place the termination screen into the microcarrier launch assembly, then place the carrier membrane dotted with gold dust particles and dried into the carrier membrane holder, mount it in the retention slot (with the side of the carrier membrane with the gold dust particles facing down), screw on the cap, and place the microcarrier launch assembly in position under the rupture disk retaining cap.
④ Place the Petri dish with young embryos on the sample holder, which is placed on the third stop counting up from the bottom of the bombardment chamber, take away the Petri dish cover, close the bombardment chamber door, evacuate to make the vacuum level reach 28 in.Hg and hold it, and then bombard.
⑤ When the vacuum is released, open the door of the bombardment chamber, put back the petri dish cover, and prepare for the next bombardment.
(6) After bombardment, the young embryos were incubated at 25°C in the dark.
5. Transformer screening
( 1 ) 16 h after bombardment, transfer the young embryos from hypertonic medium to the healing tissue induction medium containing 5 mg/L bis(propylamino)phosphine for selection, and incubate them in dark culture at 25℃.
( 2 ) The medium containing 5 mg/L bis(allylphosphine) was changed every two weeks.
( 3 ) After 4 weeks of selective culture, i.e., the second time when the medium was changed, the healing tissues originated from the same embryo were divided into 3~6 small pieces and labeled to ensure that all the healing tissues originated from the same embryo were together.
( 4 ) After 6 weeks of selective culture, i.e., the third medium change, transfer the remaining healthy healing tissues to the transition medium containing 1 mg/L bis(propylamino)phosphine, and cultivate them under low light at 25℃, i.e., put the petri dishes into a lighted tissue culture room, and cover each petri dish with a piece of thin paper.
6. Regeneration of transgenic plants
( 1 ) After two weeks of culture on transition medium, transfer the tissues originating from the same young embryo to regeneration medium (Fig. 8.2). The medium was prepared without any growth regulator and contained only 1 mg/L bis(propylamino)phosphine, using a petri dish with a large depth. Succession was performed every 2 weeks with the same medium until no new regenerated seedlings grew.
( 2 ) When the shoots of the seedlings grew to 2~3 cm and roots were also formed, they were removed from the petri dishes and transferred to glass test tubes (Sigma C-5916) containing 12 ml of callus induction medium. The medium did not contain any growth regulator and contained only 1 mg/L of bis(propylamino)phosphine.
( 3 ) When the rooted seedlings reach the top of the test tube, they can be transferred to soil. The seedlings were gently removed from the test tubes with long forceps, and the tissue culture medium on the roots was rinsed with water.
( 4 ) Plant the seedlings in 5 cm diameter pots with the same barley growing medium, and cover the seedlings with a perforated plastic cup to maintain humidity until the seedlings are well established in the soil.
( 5 ) Once the seedlings are well established in the soil, the leaves can be collected and analyzed for the presence of the target gene. Bis(propylamino)phosphine selection is not like thiamphenicol selection (see Chapter 9, "Agrobacterium-mediated Barley Transformation Methods," for details), and some non-transformed plants may survive.
( 6 ) A quick and easy way to test the transformation status of regenerating plants is to use leaves for herbicide resistance testing [8]. 
7. Transient testing with a gene gun
An important use of gene gun bombardment is to perform transient analysis, which is often used to test vectors prior to stable transformation. For transient analysis, petri dishes containing young embryos are typically bombarded twice to increase the amount of DNA transformed, and it is recommended that the dish be rotated at a 90° angle between bombardments (see Note 7). Two examples of transient expression after bombardment of young embryos with gold dust particles containing the glucosinolate lyase gene (gus) and the green fluorescent protein gene (gfp) are shown in Figures 8.3 ( a ) and 8.3 ( b ), respectively. 