Wheat Flower Organ Transformation Experiment
This chapter describes a method similar to the Arabidopsis inflorescence imbibition method for the transformation of wheat floral organs. Instead of relying on tissue culture of isolated embryos, this method selects the early, middle and late stages of mononuclear microspore development (spikelet stage), and directly infiltrates cut florets with Agrobacterium tumefaciens bacterial sap carrying dual vectors of anthocyanin reporter genes and NPTII selector marker genes. This experiment is based on "A Guide to Transgenic Technology and Field Identification of Wheat Crops" (H.D. Jones, P.R. Hewlett, ed.). Hewley, eds.
Operation method
Wheat floral organ transformation
Materials and Instruments
Wheat seed Move I. Materials For more product details, please visit Aladdin Scientific website.
Spectinomycin Rifampicin
Greenhouse or walk-in grow box Orgreen fertilizer Plastic pots LB medium MS medium Anatomical mirrors Paromomycin
1. Plant material
Crocus or Chinese Spring wheat seeds were provided by the USDA National Germplasm Resources Information Center (http: // www . ars- grin . gov / npgs /). Crocus is a red durum Canadian spring wheat germplasm line [ 7 ], and Chinese Spring wheat is commonly used for genetic studies (see Note 2).
2. Growing facilities
(1) A temperature-controlled greenhouse or walk-in grow box
(2) A standardized potting medium such as peat soil #2 (Fafard #2) (Knoxville Seed and Greenhouse)
(3) General Purpose Osmocote Fertilizer 14-14-14 (Osmocote) (Hummert International)
(4) 6 in (1 in = 0.0254 m, later) plastic pots
3. Agrobacterium culture
(1) Utilize a glycerol-frozen Agrobacterium monoclone obtained by standard electrokinetic transformation, carrying Agrobacterium strains AGL1 or C58C1 with the pBECKSred plasmid vector [ 8, 9 ] (see Note 3).
(2) LB medium containing 50 μg/ml spectacularin and 50 μg/ml rifampicin (Fisher Biotech).
(3) LB agar plates [ LB solid medium: 12 g/L agar powder (Fisher), 50 μg/ml spectacularin, 50 μg/ml rifampicin ].
(4) YEP solid medium: 20 g/L of tryptone, 10 g/L of yeast powder, 50 μg/ml of grandimycin, 50 μg/ml of rifampicin, 200 μmol/L of acetosyringone (3',5'-dimethoxy-4'-hydroxyacetophenone). Acetosyringone was dissolved in dimethyl sulfoxide (DMSO) .
4. Infiltration medium and Agrobacterium treatment
(1) 1/2 MS medium (Fisher Biotech) [10]: pH 5.8, 5% sucrose (m/V), 0.04% Silwet L-77 (V/V), 0.5 mmol/L MES (2-N-morpholinoethanesulfonic acid), and 200 μmol/L acetosyringone (see Note 4) [ 11, 12].
(2) Small plastic vials that contain 250 ml of liquid and can withstand autoclaving.
(3) Small, clean plastic bags and cellophane bags for pollination.
(4) Small scissors and tweezers.
5. Sifting
(1) Dissecting mirror
(2) Commercial bleach (30%); 1000 ppm (1 ppm = 10-6, later) of cephalexin (Fisher) Alternate [13 ].
(3) Spray with 2% baromycin (m/F, Fisher) containing 0.2% (V/V) surfactant tween 20 [14].
(4) NPTII ELISA (enzyme immunoassay) kit (Agdia, Inc, Indiana) and an enzyme labeler with adjustable reading at 540 nm.
6. Southern hybridization to verify the integration of T-DNA and to detect the copy number.
See Chapter 13.
Methods
1. Wheat growing conditions
(1) The ambient temperature for growing wheat (in a greenhouse or climate chamber) should not exceed 25°C. The ideal daytime temperature is 21~25°C and the nighttime temperature is 16°C. Keep 16 h of light. The ideal daytime temperature is 21~25°C, and the nighttime temperature is 16°C, and 16 h of light is maintained.
(2) Fill 6 in pots with peat soil substrate (Fafart # 2 ) containing Osmocote 14-14-14 (Universal) fertilizer and hole-sow wheat seeds. Add additional nutrients to the substrate if necessary.
(3) Although there is no specific range of light intensity and light quality required, it should not be less than 400-500 μE.
(4) Water thoroughly at once before transforming wheat flower organs with Agrobacterium, and do not water again within 2 days after Agrobacterium transformation.
2. Cultivation of Agrobacterium and induction of Vir gene
(1) Inoculate Agrobacterium strains carrying plasmids (frozen in glycerol) into 5 ml of LB medium, add appropriate antibiotics, and incubate at a speed of about 160 r/min at (22±2 ) °C overnight.
(2) On the second day, 2.5 ml of shaken fresh strain was inoculated into 250 ml of YEP medium (containing antibiotics and 200 μmol/L acetosyringone), and incubated at 160 r/min at (22±4 )℃ until the optical density was 1.0 ( OD600 ).
3. Infiltration medium
The medium containing YEP/Agrobacterium was centrifuged at 6000 r/min for 15 min at room temperature, after which the supernatant was discarded. Gently resuspend Agrobacterium to a final concentration of 1.0 ( OD600 ) and add Silwet-77 to the permeabilization medium before treating the plants to be transformed.
4. Preparation of wheat sheaves and treatment with Agrobacterium
(1) Select wheat ears at the gestation stage (early, middle and late stages of mononuclear microspore development) for transformation. At this time, wheat ears are still wrapped in leaf sheaths and are 6-7 cm long ( Fig. 6.1, see Note 5 ).
(2) The leaf sheath is opened to reveal the developing spikelet, and the apical and internal flowers can be removed or retained due to the high sterility rate. The remaining flowers are cut slightly below the tip of the glume bundle.
(3) Mix the Agrobacterium infiltration medium and immerse the wheat ears in the medium for 1-2 min (see Note 6).
(4) Cover the soaked ears with a small transparent plastic bag, making sure that the bag maintains high humidity for 2 days.
(5) Remove the plastic bag and allow the ears to return to normal growth. After drying the Agrobacterium-treated sheaves, cover the sheaves with a cellophane bag to prevent cross-pollination between wheat plants as the plants are approaching the flowering stage, and allow the replacement seeds to set naturally (see Note 7). 
5. Screening
Before using time-consuming and laborious transgenic assays such as Southern hybridization for copy number identification, reverse transcription-polymerase chain reaction (RT-PCR) or Northern hybridization for detection of the expression of target genes, a simple set of methods is used for screening.
6. Seed visualization
(1) The seeds were visualized under a body microscope and screened for the generation with reddish embryos, using the wild type as a control (Fig. 6.2 , see Note 8).
(2) Selected transgenic seeds were shaken and washed in 30% commercial bleach solution for 30 min to kill residual Agrobacterium, and then rinsed with sterilized water three times, each time shaking and washing for 5 min, and subsequently sown in the substrate.
(3) Optional procedure: treat germinating transgenic seedlings (optional procedure for step 1 - section 3.6) with 1000 ppm concentration of cephalexin for 2 h at room temperature to kill residual Agrobacterium [13]. 
7. Screening for resistance at whole plant level using 2% balunomycin
(1) Transgenic seedlings and wild-type plants were sprayed with 2% (m/V) balunomycin [ containing 0.2% (V/V) tween surfactant ] when they reached the 3 to 4 leaf stage [14].
(2) After the plants continue to grow for 5~7 days, the resistant and sensitive strains were counted separately (see Note 9 ), and the resistant strains were selected and retained as potential transgenic positive strains.
8. ELISA screening of NPTII gene
(1) Refer to the NPTII ELISA manual (Agdia Inc.).
(2) It is very important that the NPTII standard samples in the kit be lysed with protein extracts from wild-type control plants.
(3) To avoid false positives, select more than one wild-type plant as a negative control whenever possible (see Note 10).
(4) Select healthy tissues from transgenic plants, add Agdia Protein Extract, and extract and concentrate the protein samples using a microcentrifuge tube (tissue mass: buffer volume = 1 : 5 ). Submerge the plant tissue with a 1 ml tip, grind it with a pestle and mortar, centrifuge it for 5-10 min (about 12000 r/min), and store the supernatant for later use. The above experimental procedures were carried out at 4°C.
(5) Each sample was divided into triplicates as far as possible, and one of the samples was taken out for ELISA.
(6) At the end of the ELISA, the reaction was terminated by adding 3 mol/L H2SO4, and then quantitatively detected by selecting the 450 nm wavelength on an enzyme labeling instrument. the ELISA reading was generally higher than that of the wild-type strain, which could be initially judged as a positive transformed strain.
9. Southern hybridization was used to verify the integration of T-DNA and to detect the copy number.
The genomic DNA of transgenic plants, wild-type control and plasmid was extracted separately, and after the DNA was digested completely, the enzyme sections were separated by agarose gel electrophoresis, transferred to the membrane, and finally hybridized with the complementary probes of the target genes. See Chapter 13.