Agrobacterium-mediated transformation of barley has been widely utilized over the last decade and it offers advantages over gene gun-mediated transgenic techniques in terms of efficiency and quality of transgenic plants obtained. This method can produce a large number of fertile transgenic independent lines and is therefore ideal for conducting gene function studies in cereal crops. The source of this experiment is "Experimental Guide to Transgenic Technology and Field Identification of Wheat Crops" [English] H.D. Jones P.R . Hewley, eds.
Operation method
Agrobacterium-mediated transformation of barley
Materials and Instruments
Plant Gel pBract Plasmid pSoup Plasmid Move 1. Preparation of tissue culture medium For more product details, please visit Aladdin Scientific website.
Rifampicin Kanamycin
MG L Medium
Prepare plant gel at 2 times the desired concentration in advance and autoclave (see Note 3 ). All tissue culture media should be filter-sterilized, so except for the sterile stock solution, mix the other media components in 2x the desired concentration, adjust to the appropriate pH, and then filter-sterilize (use bottle top filters, 0.22 μm microporous membranes, Millipore). Heat the 2x gel and 2x medium in a water bath to 60°C before use. The required stock solution was added to the hot medium under sterile conditions, and the medium and plant gel were mixed and poured into 9 cm Petri dishes (Sterilin ). For differentiation culture, the medium was poured into a deeper tissue culture dish (100 mm X 20 mm, Falcon).
2. Preparation of Agrobacterium rhizogenes for inoculation
( 1 ) The standard inoculum was prepared by the method described in the literature [ 2 ] with slight modification.
( 2 ) Pick a single Agrobacterium AGL1 clone containing pBract plasmid and pSoup plasmid, inoculate it into 10 ml of MG/L medium containing 25 μg/ml rifampicin and 50 μg/ml kanamycin, and incubate it for 40 h at 28℃ with 180 r/min shaking.
( 3 ) Add 10 ml of sterile 30% glycerol into the bacterial suspension, and mix upside down for several times.
( 4 ) Dispense 400 μl of standard inoculum into a 0.5 ml centrifuge tube and leave it at room temperature for 2 h. Upside down mixing was done every 30 min.
( 5 ) Store the standard inoculum at -80℃.
3. Isolation of young barley embryos
(1) Harvesting and sterilizing young embryos
(1) Harvest the ears when the embryos have grown to 1.5-2 mm in diameter (see Note 4).
② Before cutting the ears, take a seed from the center of each ear and check the size of the young embryo to ensure that it meets the requirements [Fig. 9.2 (a)~(c)].
(iii) Remove the mature seeds and remove the awns, but do not damage the hulls.
④ Sterilize the seeds with 70% ethanol for 30s, then rinse with sterile water for 3 times.
⑤ Soak the seeds in sodium hypochlorite (sodium hypochlorite solution, Fluka 71696) in a 50:50 ratio of water for 4 min.
⑥ Wash the seeds 4 times with sterile water, pour off the water but keep the seeds moist, and place them in sterile screw-top vials for use.
(2) Isolation of young embryos and removal of embryonic axes
① All operations were performed on a sterile bench.
② About 20 sterilized seeds were taken at a time and placed on the sterile blue or black backing of a dissecting microscope.
③ Hold the seeds in place with one forceps and peel out the young embryos with the other forceps, removing the embryonic axes (see Note 5) [Fig. 9. 2 ( c )~(d)].
④ Place the young embryo, shield side up, on the healing tissue induction medium.
⑤ Place 25 young embryos in each 9 cm diameter petri dish and prepare for inoculation with Agrobacterium. 
4. Agrobacterium preparation, inoculation and co-culture
( 1 ) Add Agrobacterium standard inoculum into 10 ml MG/L liquid medium without antibiotics and incubate at 180 r/min at 28℃ overnight (about 20 h).
( 2 ) Inoculate the young embryos with Agrobacterium culture solution.
( 3 ) Apply a small amount of Agrobacterium onto each embryo with a 200 μl pipette gun so that it just covers the surface of the embryo.
( 4 ) After treating 25 embryos in a petri dish, tilt the dish so that the excess Agrobacterium on the embryos will flow out.
( 5 ) Inoculate up to 2 petri dishes of young embryos at a time and then carefully transfer the young embryos to another fresh induction medium with the shield facing down.
( 6 ) Take care not to carry excess medium or Agrobacterium into the new medium. If necessary, gently swipe the embryos across the medium to remove excess Agrobacterium before transferring to the new medium.
( 7 ) Seal the petri dish with medical tape and incubate at 23~24°C for 3 days. It is convenient to isolate the young embryos on the first day and inoculate them on the second day, but of course it is possible to inoculate them on the same day of isolation (see Note 6).
5. Selection of Transformants
( 1 ) After 3 days of co-cultivation, transfer the young embryos to healing tissue induction medium containing thiamphenicol for selective culture and trimethoprim for inhibition of Agrobacterium (see Note 7).
( 2 ) The young embryos are incubated shield-side down at 23-24°C in the dark. This step is called selection 1.
( 3 ) After 2 weeks, the embryos are transferred to a new selection medium (selection 2). The same young embryo and its resulting healing tissue are transferred as a whole and not separated.
( 4 ) After 2 weeks of further incubation, the embryo is transferred to a new selection medium (Option 3) (see Note 8), during which time the guaiac will detach from the embryo, and is placed around that embryo and labeled so that the growth of all the guaiac tissues originating from the same young embryo can be tracked.
( 5 ) Reduce the number of embryos per dish because the resistant healing wounds of embryo origin will begin to grow rapidly.
( 6 ) After 6 weeks of selective culturing on the healing tissue induction medium, transfer the healing tissues to the transition medium containing thaumatin and terramycin. The culture was incubated at 24℃ under low light for 2 weeks, i.e., the petri dishes were put into a lighted tissue culture room and covered with a thin sheet of paper to obtain a darker light. After these two weeks of incubation, the transformed healing wounds were clearly distinguishable and began to show green areas and small shoots. The non-transformed healing wounds hardly differentiated on the medium containing thaumatin.
6. Regeneration of transgenic plants
( 1 ) After 2 weeks of culture on transition medium, the embryonic tissues were finally transferred to regeneration medium in tall Petri dishes without any growth regulator but containing the same concentration of thiomectin and terramycin.
( 2 ) Since transformed healing wounds grow very fast, the number of healing wounds in each medium needs to be further reduced.
( 3 ) The morphology of the guaiac in a typical regeneration is shown in Fig. 9.3 ( a ) and Fig. 9.3 ( b ). Regenerated healing wounds from the same source of young embryos should be placed together.
( 4 ) When the shoots of the seedlings have grown to 2~3 cm and roots have also formed, they are removed from the petri dishes and transferred to glass test tubes (Sigma C-5916) containing 12 ml of healing tissue induction medium without dicamba or other growth regulators, but still with the same concentration of chaotropic acid and trimethoprim.
( 5 ) Plants transferred to test tubes for growth are shown in Fig. 9.3 ( c ) and Fig. 9.3 ( d ). The transgenic plants were able to form strong roots quickly in the rooting medium containing thaumatin.
( 6 ) Plants that form a strong root system in medium containing thiamphenicol tend to be successfully transformed, and no escaped or non-transformed plants will survive using this screening method (see Note 9). 
( 7 ) When rooted plants reach the top of the test tube, they can be transferred to soil.
( 8 ) The seedlings are gently removed from the test tubes with long forceps, and the rooted tissue culture medium is rinsed off with water and planted in 5-cm-diameter pots containing the same barley growth medium species.
( 9 ) Seedlings may be covered individually with small perforated plastic cups or the entire pot may be covered with a lid to maintain humidity until the seedlings are well established in the soil.
( 10 ) Once the seedlings are well established in the soil, the leaves can be taken and analyzed for the presence of the target gene. Figure 9. 4 shows the expression of the gus gene in the leaves of transgenic plants. 