Three wheat NILs of CB037A, CB037B, and CB037C were identified from the original CB037 and provided by Prof. Yueming Yan at Capital Normal University, which were found to be different in glutenin composition (Wang et al. 2016). Two wheat NILs of Pst-resistant line CB037-PstR and Pst-susceptible line CB037-PstS were identified from the original CB037 and provided by Prof. Jiajie Wu at Shandong Agricultural University (Zheng et al. 2020), which were found to be different in stripe rust resistance. Spring wheat line Chinese Spring (CS) which was preserved in our laboratory. The wheat materials were planted by three replications in the February of 2018 at the experimental station of Institute of Crop Sciences (ICS), Chinese Academy of Agricultural Sciences (CAAS), Beijing, for anther, immature embryo, and mature embryo culture, and PM and stripe rust resistance identification.
Expression vector pWMB110-GUS was constructed by our laboratory (Figure S1). Agrobacterium strain C58C1 was kindly provided by Prof. Tom Clemente at University of Nebraska-Lincoln, USA.
Genomic in situ hybridization
Wheat roots were collected from the germinating seeds on an absorbent filter paper for 2–3 d at 25 ℃ in darkness. The root tips were treated with nitrous oxide for 2 h on 0.1 Mpa, and then fixed for 5 min with 90% glacial acetic acid. Chromosomes were prepared according to a previously described method (Yuan et al. 2014). The total genomic DNA of D. villosum labeled with fuorescein-12-dUTP using the nick translation method and using genomic DNA of CS as a block was employed as a probe for genomic in situ hybridization (GISH) to identify the chromosome constitution of the three NILs of CB037 following the methods described previously (Yuan et al. 2014; Guo et al. 2016). Slides were visualized using an Olympus BX-51 fluorescence microscope (Olympus Corporation, Japan) and photographed using a charge-coupled device system.
Powdery Mildew And Stripe Rust Resistance Test
For PM resistance test, the wheat plants at the tillering stage were inoculated with Blumeria graminis (Bgt) mixture collected from the infected wheat seedlings grown in artificial conditions. Two weeks after inoculation, disease symptoms of the wheat plants were investigated. In immune type there were no lesions and Bgt pathogens on the leaves, and in susceptibility type there were continuous lesions and a serious covering of Bgt pathogens on the leaves (Xiao et al. 2013).
For stripe rust resistance test, the wheat plants at the seedling stage were inoculated with Puccinia striiformis f. sp. tritici (Pst) and talc powder which were mixed in 1:10 ratio using a small brush to gently smear on the area of one leaf. Two weeks after inoculation, a large number of spores were found in the inoculated leaf area of the susceptible plants, while no lesions and Pst spores were observed in the inoculated leaf area of the resistant plants (Zheng et al. 2020).
The wheat spikes with two stem nodes and leaves in which the development of the pollen cells was at uni-nucleus middle-late stage after mitosis were collected in a plastic bag and pretreated under 4 ℃ for 3 d in a refrigerator. Then, the leaves and second stem nodes were removed; the remaining tissues consisting of spike, sheath and the first stem node were carefully wiped with 70% alcohol for surface disinfection; the sheath and glumes were peeled off by tweezers; the anthers were inoculate on W14 medium (pH 6.0) containing 2.0 mg/l 2,4-D, 0.5 mg/l KT, 100 g/l sucrose, and 2.4 g/l gelrite (Zhang et al. 2018). The isolated tissues were firstly cultured at 30 ℃ for 3 d, and then at 28 ℃ for 30–45 d in darkness for callus induction. The calluses were moved onto FHCK medium (basic MS medium containing 30 g/l sucrose and 2.4 g/l gelrite, pH 6.0) for 20–25 d at 25 ℃ for shoot regeneration under a 16 h-light/8 h-dark photoperiod with a 100 µmol/m− 2/s− 1 photosynthetic photon flux density (She et al. 2013; Zhang et al. 2018). All media were autoclaved at 121°C for 15 min. Each experiment was duplicated at least three times.
Immature embryos culture
Wheat seeds were planted in a growth chamber with a temperature regime of 25 ℃-day/20 ℃-night, a photoperiod of 16 h-light/8 h-dark, a photosynthetic photon flux density of 300 µmol/m− 2/s− 1, and a relative humidity of 45%. Wheat immature grains at 13–14 d post anthesis (DPA) were collected, surface sterilized with 70% ethanol for 1 min and 15% sodium hypochlorite for 10 min in order, and rinsed for four times with sterile water. Wheat immature embryos of 1.2–1.5 mm in size were aseptically excised from the sterilized grains after the embryogenic axes were carefully removed, and then incubated with scutellum upwards on callus induction medium (CIM containing MS inorganic, 10.0 mg/l vitamin B1, 150 mg/l asparagine, 2.0 mg/l 2,4-D, 30 g/l sucrose, and 2.4 g/l gelrite, pH 6.0) in 90-mm of diameter petri dishes in darkness at 25 ℃ for 14–21 d for callus induction (She et al. 2013; Zhang et al. 2018). In total, 150 immature embryos approximately from each wheat line were incubated on three petri dishes for duplication. The embryonic calluses were moved onto FHCK medium for another 14–21 d at 25 ℃ in light conditions as aforementioned description for shoot differentiation. Three duplicates at least were set up for this experiment.
Mature embryos culture
Mature wheat seeds were sterilized with 70% ethanol for 10 min and 25% sodium hypochlorite for 25 min, and then soaked in sterile water overnight at 25°C in darkness after being rinsed with sterile water for four times. The slightly germinated seeds were sterilized again with 25% sodium hypochlorite for 15 min and then rinsed with sterile water for four times (Yin et al. 2011; Zhang et al. 2018). The mature embryos were scraped into small pieces on seeds with a sharp knife and cultured on Adi medium (4.3 g/l MS inorganic, 30 g/l maltose, 1.0 g/l casein hydrolysate, 0.35 g/l myoinositol, 0.69 g/l proline, 1.0 mg/l VB1, 2.5 mg/l dicamba, 0.5 mg/l 2,4-D, 3.5 g/l gelrite, pH 6.0) at 25 ℃ in darkness for 7 d for initial callus induction, in which every two embryos were scraped and cultured together. Then, the initiative calluses were moved onto fresh Adi medium for 3 weeks at 25 ℃ in darkness. Finally, the embryonic calluses were cultured on FHCK medium for 2 weeks at 25 ℃ in light conditions as aforementioned described for shoot regeneration. Each experiment was duplicated at least three times.
Agrobacterium-mediated transformation of wheat immature embryos
Wheat heads grown in the growth chamber were sampled at 14–15 DPA. The immature grains were carefully collected, then surface sterilized with ethanol and sodium hypochlorite in order, and finally rinsed with sterile water in aseptic conditions by the aforementioned procedure. A proprietary Agrobacterium mediated transformation system of wheat developed by Japan Tobacco Company (Ishida et al. 2015) was applied with a slight modification. In brief, immature wheat embryos of 2.0-2.5 mm in size were cautiously isolated from the sterilized grains aseptically under a stereoscopic microscope, incubated with the Agrobacterium strain C58C1 harboring pWMB110-GUS expression vector for 5 min on WLS-inf medium at room temperature, and co-cultivated with the scutellum facing upwards for 2 d on WLS-AS medium at 23°C in darkness. After co-cultivation, embryonic axes were removed and the remaining scutella were transferred onto WLS-Res medium. After a delay culture for 5 d, the tissues were transferred onto callus induction (WLS-P5) medium. Two weeks later, the survived calli were sliced vertically into halves and evenly cultured on WLS-P10 medium for 3 weeks in darkness. Embryogenic calli were then moved onto LSZ-P5 medium at 25 ℃ in light conditions as aforementioned description. Regenerated shoots were transferred into cups filled with MSF-P5 medium for shoot elongation and root formation. Plantlets with well-developed root systems were transplanted into pots and cultivated in the growth chamber as aforementioned described.
Agrobacterium-mediated transformation of wheat mature embryos
Wheat mature embryo tissues were prepared by the description mentioned above. The initiative calli produced from the fine wheat mature embryo pieces which were cultured on Adi medium for 7 d were incubated with the Agrobacterium harboring the pWMB110-GUS vector for 30 min in WLS-inf medium at room temperature, and co-cultivated for 3 d on a sterile filter paper at 23°C in darkness. Then, the infected calli were transferred onto Adi-Res medium (basic Adi medium containing 0.25 g/l carbenicillin and 1.0 g/l vitamin C, 0.85 mg/l AgNO3, pH 6.0). After 5 d at 25 ℃ under darkness, the tissues were transferred onto callus selection medium (Adi-P5: basic Adi medium containing 0.25 g/l carbenicillin, 1.0 g/l vitamin C, 0.85 mg/l AgNO3, and 5 mg/l phosphinothricin (PPT), pH 6.0). After two weeks, callus cultures were placed on Adi-P10 medium (basic Adi medium containing 0.25 g/l carbenicillin, 1.0 g/l vitamin C, 0.85 mg/l AgNO3 and 10 mg/l PPT, pH 6.0) for three weeks at 25 ℃ in darkness. Embryogenic calli were then cultured for differentiation on FHP5 medium (basic FHCK medium containing 0.25 g/l carbenicillin and 5 mg/l PPT, pH 6.0) at 25 ℃ in aforementioned light conditions. Regenerated shoots were transferred into cups filled with FHP5 medium for rooting. Plantlets with well-grown shoots and roots were transplanted into pots and cultivated in the growth chamber as aforementioned described.
Detection of transgenic wheat plants by PCR
Genomic DNA was extracted from the leaves of the putative transgenic wheat plants using a NuClean PlantGen DNA kit (CWBIO, China). The presence of the GUS gene in the transgenic plants was tested by an amplification fragment of 898 bp using the specific PCR primer pair 5’-GACCACCAGTGCAAGAACCCTC-3’ and 5’-ATCCACGACCGACACTTTCACG-3’. The presence of the bar gene in the transgenic plants was examined by an amplification fragment of 429 bp using the specific PCR primer pair 5’-ACCATCGTCAACCACTACATCG-3’ and 5’-GCTGCCAGAAACCACGTCATG-3’.
Histochemical staining and Quickstix stripe detection of transgenic wheat plants
Histochemical staining analysis for GUS expression was conducted by a described method (Jefferson et al. 1987). The young leaves collected from the putative transgenic wheat plants were immersed directly in 0.1 M NaPO4 buffer (pH 7.0) containing Na2-EDTA 10 mM, ferricyanide 0.2 mM, ferrocyanide 0.2 mM, X-gluc 0.8 g/l, methanol 20%, and Triton-100 0.5%, and incubated overnight at 37°C. Additionally, a QuickStix Kit (EnviroLogix, LibertyLink) was applied to detect the Bar protein in putative transgenic wheat plants by the manufacturer’s instructions.
Data statistical analysis
For tissue culture, the numbers of primary calli, embryonic calli, and green plantlets were counted in each experiment, and then the data was sorted by Microsoft Excel. The regeneration efficiencies of wheat immature embryos and mature embryos for the three near-isogenic lines were compared by calculating embryogenic callus induction rate (number of embryogenic calli/number of incubated immature or mature embryos × 100%), differentiation callus rate (number of the calli showing green shoots/number of incubated immature or mature embryos × 100%), shoot production rate (number of regeneration shoots/number of incubated immature or mature embryos × 100%).