Experiments on efficient Agrobacterium transformation methods in wheat mediated by in situ plant inoculation
Experiments on efficient Agrobacterium transformation methods in wheat mediated by in situ plant inoculation
This chapter details a reproducible method of Agrobacterium-mediated transformation of spring wheat by direct inoculation of Agrobacterium sap into immature seeds of wheat, which has been patented WOOO/63398 [1]. Its transformation efficiency ranges from 1% to 30%, and an average transformation efficiency of at least 5% is usually obtained. The overall performance of the regenerated plants obtained from the transformation was normal. 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, ed.
Operation method
Efficient Agrobacterium transformation of wheat mediated by in situ plant inoculation
Materials and Instruments
Slow-release fertilizer Acetosyringone Move I. Materials For more product details, please visit Aladdin Scientific website.
Potager Plant Supporter LB Medium Universal Test Tube YEP Solid Medium TSIM Medium
1. Growth of the parent plant
( 1 ) Parent plants were planted in 12 , 7F (13 cm diameter) pots with 4 plants per pot.
( 2 ) Use Levingtons M2 culture soil and apply 5 g/L of slow-release fertilizer (15 : 9 : 9+ 3 MgO + TE; release cycle up to 5-6 months) with a thin layer of vermiculite on the surface of the medium.
( 3 ) Teku plant supports (LBS Horticulture Colne Lancashire, UK) were used to support the plants.
( 4 ) Trays were lined with Aquamat ( LBS Horticulture) and 6 cans were placed in each tray (deep gravel trays 30 cm wide at the bottom and 50 cm long).
( 5 ) Watering was controlled by Octamitters.
( 6 ) The growth tank used was a Conviron PGV/PGW 36.
2. Preparation of Agrobacterium glycerolatum in small quantities
The following materials need to be prepared:
( 1 ) LB medium (Sigma- Aldrich)
( 2 ) Universal test tubes (25 mL, Sterilin)
( 3 ) 85 : 15 LB medium/glycerol solution (Sigma- Aldrich)
3. Agrobacterium preparation and inoculation
The following materials need to be prepared:
( 1 ) YEP solid medium: 5 g/L yeast extract, 10 g/L peptone, 5 g/L NaCl, 15 g/L agar, pH 6.8 (all of the above were purchased from Sigma- Aldrich).
( 2 ) TSIM medium: 4.4 g/L MS salts and vitamins (MP Biomedicals), 100 mg/L inositol, 10 g/L sucrose, 500 mg/L MES buffer (purchased from Sigma-Aldrich), pH 5.2, filtered for sterilization.
( 3 ) Acetosyringone (3 ,5 -diphenoxy- 4- hydroxyacetophenone) ( Sigma- Aldrich), 0.1 mol/L aqueous solution.
( 4 ) 25 μl to 2.5 ml Hamilton reusable syringe dispenser (Fig. 7.1). 
( 5 ) Hamilton syringe (50 μl) with needle.
( 6 ) Measuring cylinder (500 ml)
( 7 ) Glass rod or similar support
( 8 ) Transparent plastic bag (455 mmx 600 mm)
( 9 ) Sanyo Multi-functional Environmental Monitoring Case
4. Tissue culture
The following materials are required:
( 1 ) Domestos Bleach (Lever Bros)
( 2 ) Ethanol solution (70% V/V): prepared in deionized water
( 3 ) Sterile deionized water
( 4 ) Scalpel with #15 blade (Swann-Morton, Sheffield, UK)
( 5 ) W4 medium: 4.4 g/L MS salts and vitamins (MP Biomedicals) , 20 g/L sucrose (Merck KGaA, Germany) , 2 mg/L 2,4-D, sodium salt (Sigma- Aldrich) , 500 mg/L glutamine (Sigma-Aldrich) , 100 mg/L Casein hydrolysate (Sigma-Aldrich) , 6 g/L type 1-A agarose (Sigma-Aldrich) , 150 mg/L ticarcillin disodium/potassium clavulanate (Melford Laborato ries Ipswich, UK), pH UK), pH 5.8. Table 7.1 lists the preparation details of the media additives. 
( 6 ) W425G medium: The rest of the medium was the same as W4 medium with the addition of 25 mg/L G418, bisulfate ( Sigma-Aldrich ).
( 7 ) MRM medium: 4.4 g/L MS salts and vitamins (MP Biomedicals), 20 g/L sucrose (Merck KGaA, Germany), 6 g/L type 1-A agarose (Sigma-Aldrich), 2 mg/L agonin (SigmaAldrich), pH 5.8.
( 8 ) MRM25G medium: the rest was the same as MRM medium, with the addition of 25 mg/L G418, bisulfate (Sigma-Aldrich).
( 9 ) Beatson wide-mouth flasks (250 ml, Richardsons, Leicester, UK) were capped with the bottom of a covered Petri dish and sealed with 25 mm 3M Micropore's tape.
( 10 ) MS20 medium: 4.4 g/L MS salts and vitamins (as before), 20 g/L sucrose (Merck KGaA, Germany) , 7 g/L agar (Sigma- Aldrich) tested in plant cell culture, pH 5.8 .
( 11 ) Sanyo multifunctional environmental monitoring kit.
5 Growth of transgenic seedlings
The following materials are required:
( 1 ) Jiffy 7 peat pellets (Jiffy Products, Norway)
( 2 ) Seeding trays and corresponding 24-hole hole trays (supplier of choice)
( 3 ) Incubator suitable for the size of the seeding tray (optional)
6. Growth of transgenic plants
The following materials are required:
( 1 ) 12F (12 cm diameter) pots and pans, one plant per pot.
( 2 ) Cultivate the plants with a mixture of Levingtons M2 and apply 5 g/L of slow-release fertilizer for OGL (same as before)
( 3 ) Use Pea sticks/split canes as support.
( 4 ) Microporous bread bags (Focus Packaging) were used for bagging.
II. METHODS
1. Growth of mother plant
( 1 ) Sow seeds into M2 fertilizer mix with slow-release fertilizer (as above) at a depth of 2-3 cm ( 4 seeds per can).
( 2 ) Place a thin layer of vermiculite on the surface of the pots to maintain the quality of the surface fertilizer mix. Place one Teku plant supporter per pot.
( 3 ) Place the pots in a Conviron growth chamber under incubation conditions of 20°C during the day and 15°C at night, with a 16 h photoperiod and 400 μE/( m2-s ) light (see Note 1).
2. Harvesting
( 1 ) Harvest the tillers 16 to 18 days after flowering, when the embryo grows to 1 mm in size. Cut the tillers from the base of the plant (see Note 2).
( 2 ) Discard the bottom leaves and keep the blade.
( 3 ) Immediately place the tiller in a 500 ml measuring cylinder of water, trimming the length of the tiller so that the raphe is at the top of the cylinder.
3. Threshing
( 1 ) Remove 2-3 spikelets from the bottom of each spike.
( 2 ) Select the first and second flowers from the bottom up (Fig. 7.2) and carefully remove the glumes and lemmas from the flowers so that only the immature seeds of these two flowers are exposed.
( 3 ) Cut away the residual tissue that interferes with inoculation, i.e., the lemmas/glumes and awns of the unmoved flowers (e.g., the third and fourth flowers, etc.), never damaging the exposed seeds, which can lead to fungal contamination during co-culture.
( 4 ) Brush the spike upwards with a soft brush to remove anthers, which may also otherwise cause fungal growth.
( 5 ) Wipe the leaves with a damp paper towel.
( 6 ) Lightly spray spikes with 70% (V/V) ethanol to dry them before inoculation (see Note 3).
4. Preparation of Agrobacterium plates
( 1 ) Prepare a small amount of Agrobacterium glycerolatum containing the plasmid.
( 2 ) Pick a single clone from the mother plate and incubate it in LB medium with appropriate antibiotics [ 16 ] at 28℃ overnight with vigorous shaking. Remove 1 ml into an Eppendorf tube, centrifuge at 10000 g for 30 s. Remove the supernatant and resuspend the cells in 1 ml of LB with 15% (m/V) glycerol. Dispense into small portions and store at -80°C. Use a fresh portion of Agrobacterium glycerol for each inoculation and discard after use.
( 3 ) Prior to the inoculation experiment, line a YEP plate containing the appropriate antibiotic with the preserved Agrobacterium glycerophilus so that the entire plate is covered with spots.
( 4 ) Seal the plate and incubate at 28℃ for 1 day or at room temperature for 3 days. 
5. Agrobacterium resuspension
( 1 ) After the wheat ears for inoculation are prepared, start preparing the suspension of Agrobacterium.
( 2 ) Wipe and cauterize a flathead spatula with ethanol to cool it down sufficiently.
( 3 ) Gently scrape the surface of the plate with the spatula to collect a sufficient amount of bacteria, and then transfer to a 25 ml Universal tube containing 10 ml TSIM and 400 μmol/L acetosyringone. Avoid taking the adhesive on the plate with you as this will interfere with the inoculation process.
( 4 ) Resuspend the bacteria by blowing repeatedly with a 1 ml Gilson pipette gun until a homogeneous suspension is achieved.
6. Inoculation
( 1 ) Sterilize the syringe 4 to 5 times with 70% (V/V) ethanol and then rinse 4 to 5 times with sterile water. Make sure the needle hole is facing towards you.
( 2 ) Aspirate Agrobacterium suspension with a syringe.
( 3 ) Hold the spike in your hand so that the tip of the needle can reach the embryo of the seed. Place the needle into the spike so that the tip of the needle is approximately at the junction of the endosperm and the shield (Fig. 7.3). Slight damage to the shield will have no effect and may serve as evidence of proper inoculation.
( 4 ) Inject 1 μl of suspension at a time by slowly pressing the button. 50 μl syringe, 1 μl of suspension per button press.
( 5 ) Repeat the process for all exposed seeds.
( 6 ) After all spikes have been inoculated, place a support (e.g., a glass rod) in the measuring cylinder, then cover with a clear plastic bag and seal at the bottom. Allow to grow for 2 to 3 days at 22°C, 16 h photoperiod, and 40 μE/( m2- s) light (see Note 4 ).
( 7 ) Wash the syringe with 70% (V/V) ethanol 4~5 times, rinse with sterile water 4~5 times, wash with 70% ethanol once more, and then air dry. 
7. Isolation
( 1 ) After 2~3 days of inoculation, remove the seeds from the spike and put them into a suitable covered container.
( 2 ) Surface sterilization of seeds: 70% (V/V) ethanol for 1 min, 20% (V/V) Domestos bleach solution for 25 min with agitation.
( 3 ) Wash thoroughly in a strainer with sterile deionized water.
( 4 ) Separate the embryos and place them in W4 medium, shield up, 25 per dish (see Note 5).
( 5 ) Seal the plate with Parafilm and incubate at 28℃, 16 h photoperiod and 80 μE/( m2-s ) light for 5 days.
8. Tissue culture
( 1 ) After 5 days, remove the embryonic axes from the shields (see Note 6) and transfer to fresh W4 medium (see Note 7).
( 2 ) After 12 days of culture, the healing tissues were transferred to selection medium (W4 25G ). Cultivate at 25°C, 16 h photoperiod, and 80 μE/( m2-s ) illumination for 2 weeks (the next operations are performed at this temperature and illumination).
( 3 ) After 2 weeks of selection, the healing tissues were divided into small pieces for better access to the selection medium and then transferred to fresh W4 25G medium.
( 4 ) After 2 weeks of continued selection culture, the viable embryonic healing wounds are transferred to regeneration medium (MRM 25 G) containing selection reagents. After 2 weeks, another transfer is made to transfer the healing wounds that have not yet developed into buds to fresh MRM 25G medium.
( 5 ) Transfer the regenerated buds to Beatson tanks containing MS20 and allow them to incubate at 25°C (see Note 8).
( 6 ) Allow the buds to grow for about 1 month, during which time the leaves and roots should be fully developed.
9. Soil transplanting and seedling refining
( 1 ) Moisten Jiffy7 peat pellets with tap water and place in 24-well hole trays, placing the trays in a growing box.
( 2 ) Remove the seedlings from the Beatson tanks, avoiding agar on the roots.
( 3 ) Separate clumps of seedlings until each seedling has only one bud.
( 4 ) Transfer seedlings to Jiffy 7 peat pellets, making sure that all roots are Jiffy encapsulated so that the roots have access to the peat. Remove dead and dying leaves. Trim growing leaves to 5-10 cm. Sprinkle with water before putting the lid back on the terrarium (see Note 9).
( 5 ) After 2 weeks at 20°C, plants can be used for PCR testing and potted if desired (see Note 10).
( 6 ) For maximum yield, seedlings can be transferred to 12F pots with M2 culture soil (see above), one seedling per pot. Do not remove the netting from the outside of the Jiffy peat pellets to keep the pellets intact during the transfer of seedlings to soil.
( 7 ) Bag flowering plants to minimize pollen dispersal.