Plant material
Seeds of the spring wheat (Triticum aestivum L.) cv ‘Fielder’ were sown at weekly intervals in a mixture of peat and sand (85% fine grade peat, 15% washed grit, 4 kg m−3 maglime, 2.7 kg m−3 Osmocote (3–4 months), 1 kg m−3 PG Mix 14-16-18 + Te 0.02% and wetting agent). They were initially sown in 5 cm diameter pots and after approximately 4 weeks the germinated plants were transferred into 13 cm diameter pots containing John Innes Cereal Mixture (40% medium grade peat, 40% sterilised loam (soil), 20% washed horticultural grit, 3 kg m−3 maglime, 1.3 kg m−3 PG mix 14-16-18 + Te base fertiliser, 1 kg m−3 Osmocote mini 16-8-11 2 mg + Te 0.02%, and wetting agent) for continued development. Plants were grown in controlled growth chambers (Conviron Europe Ltd) at 20 ± 1 °C day and 15 ± 1 °C night temperatures, 70% humidity with light levels of 800 μmol m−2 s−1 provided by fluorescent tubes and tungsten lighting. At any stage of growth, the donor plants were not sprayed with insecticides or fungicides. In addition, particular care was taken to restrict unnecessary staff access to the controlled environment rooms, hairnets and designated lab coats were maintained in a freezer (− 20 °C) to reduce the risk of spreading pathogens.
Immature embryos isolation
Wheat spikes were collected approximately 14 days post anthesis (dpa), when the immature embryos (IE) were 1–1.5 mm in diameter (Fig. 1c, d) and early milk stage GS73 [16]. Kernels from floret 1 and 2 on central spikelet (Fig. 1a, b) were used for transformation. The awns were cut off the ears approximately 3–5 mm from the grain. The seed coat can be removed but this was not essential unless contamination problems are encountered. The immature grains were separated from the ear and placed in a 150 mL Sterilin jar. Within a laminar airflow cabinet under aseptic conditions, the grains were surface sterilised using 70% ethanol (v/v) for 1 min, given 1 rinse with sterile distilled water, followed by 7 min in 10% (v/v) sodium hypochlorite (Fluka 71696). The grains were then washed 3 times with sterile distilled water.
All subsequent operations were performed under sterile conditions in a laminar flow hood. Embryos were isolated from the immature grains using fine forceps under a dissecting microscope. Approximately 100 embryos were put into two 1.7 mL Eppendorf tubes (Fig. 1e) containing 1 mL wheat inoculation medium (WIM) modified from [11] containing 0.44 mg L−1 Murashige and Skoog (MS) [17] plant salt base (Duchefa M0222), 10 g L−1 glucose, 0.5 g L−1 2-(N-morpholino) ethanesulfonic acid (MES), 0.05% Silwet L-77 and 100 µM Acetosyringone (AS) added fresh just before use.
Construct assembly
To create a pGreen [18] based, Golden Gate cloneable vector compatible with the Modular Cloning (MoClo) system a 799 bp fragment containing the LB and a LacZ Golden Gate cassette was isolated from pAGM8031 (Addgene 48037) using restriction enzymes PshAI/PmeI, and cloned into pGreen II 0000 at the HpaI/StuI sites using blunt end ligation. This Level 2 binary vector was deemed pGoldenGreenGate-M (pGGG-M) (Fig. 2a). A reporter pGGG vector was made, for wheat transformation, which contained the hygromycin resistance gene (Hpt) and Cat1 intron driven by the rice actin1 promoter, and the β-glucuronidase gene with 2 introns (GUS2Int) driven by the rice ubiquitin promoter (Fig. 2b). Briefly, the Level 1 constructs pICH47802-RActpro::HptInt::NosT (selectable maker) and pICH47742-RUbipro::GUS2int::NosT (GUS Reporter) were cloned into the binary Level 2 vector pGGG-M using standard Golden Gate MoClo assembly [19].
Preparation of Agrobacterium for transformation
The hypervirulent Agrobacterium tumefaciens strain AGL1 [20] was used in all plant transformation experiments. Vectors were electroporated into Agrobacterium AGL1 competent cells as previously described [2], when pGreenII [18] derivatives were used i.e. pBRACT [21] or pGGG they were co-electroporated with the helper plasmid pSoup [18] or its derivatives pAL154 contained the 15 kb Komari fragment or pAL155 with an additional VirG gene.
Single colonies of Agrobacterium AGL1, which contain the desired vector, were inoculated into 10 mL of LB [22] liquid medium containing appropriate antibiotics and incubated at 28 °C, shaken at 200 rpm for ~ 65 h. A modified method of Tingay et al. [23] to prepare Agrobacterium standard inoculums for transformation was used as previously described by Bartlett et al. [2]. Equal quantities of 30% sterile glycerol and the Agrobacterium culture were mixed by inverting and aliquots of 400 μL in 0.5 mL Eppendorf tubes were made. The aliquots of standard inoculums were frozen at − 80 °C and stored until required.
The day before wheat transformation a single 400 μL standard inoculum was used to inoculate 10 mL of liquid MG/L [24] (5 g L−1 mannitol, 5 g L−1 tryptone, 2.5 g L−1 yeast, 100 mg L−1 NaCl, 1 g L−1 Glutamic acid, 250 mg L−1 KH2PO4, 100 mg L−1 MgSO4, 1 μg L−1 Biotin. final pH = 7) medium without antibiotics and incubated at 28 °C shaken at 200 rpm overnight (~ 16 h). On the day of transformation, the bacteria were pelleted by centrifugation in a 50 mL Falcon tube at 3100 rpm for 10 min at 24 °C. The supernatant was discarded, and the cells resuspended gently in 10 mL wheat inoculation medium (WIM) to an optical density of 0.5 OD (600 nm) and 100 µM AS added. The culture was incubated at room temperature with gentle agitation (80 rpm) for 4–6 h in the dark.
Inoculation with Agrobacterium and co-cultivation
The isolated embryos were placed into fresh WIM medium prior to centrifugation at 14,000 rpm at 4 °C for 10 min [25]. WIM was removed with a pipette and after 1 mL Agrobacterium solution added, the tubes were inverted frequently for 30 s and incubated at room temperature for at least 20 min. After the incubation period, the Agrobacterium suspension was poured with the embryos into a 50-mm diameter Petri plate and the Agrobacterium suspension was removed with a pipette. The embryos were transferred, scutellum side up, to the co-cultivation medium which consisted of WIM supplemented with 100 μM AS, 5 µM AgNO3, 1.25 mg L−1 CuSO4·5H2O and 8 g L−1 agarose [11]. Twenty-five embryos were placed in each 90 mm single vent Petri plate (Thermo Scientific No 101R20) and incubated at 24 ± 1 °C in the dark for 3 days co-cultivation (Fig. 1f).
Throughout the tissue culture process, all solid media components, except for the gelling agent, were prepared as a double-concentrate and filter-sterilised. The gelling agents were prepared as a double concentrate in water and sterilised by autoclave. After autoclaving the gelling agents (2×) were maintained at 60 °C and the filter-sterilised media components (2×) were warmed to 60 °C prior to mixing both and pouring. The phytohormones and antibiotics were added as filter sterilised stocks just before pouring.
Resting period, callus induction and selection of transformed material
After 3 days’ co-cultivation, the embryogenic axes were excised from the embryos using forceps (Fig. 1g). The embryos were transferred to the fresh callus induction plates (WCI) based on the media described in [26] but containing 2 mg L−1 Picloram (Sigma-P5575), 0.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D), 160 mg L−1 Timentin and 5 mg L−1 agarose and incubated at 24 ± 1 °C in the dark for 5 days. Timentin was added to control Agrobacterium during the resting period. The embryos were transferred, scutellum side up, to fresh WCI plates as above with 15 mg mL−1 Hygromycin and incubated at 24 ± 1 °C in the dark for 2 weeks. This transfer is referred to as Selection 1. The calli were split at the next transfer into clumps of approximately 4 mm−2, callus pieces derived from each single embryo were labelled to keep track of their origin. The calli were transferred to fresh selection plates (WCI) as above, but with 30 mg L−1 Hygromycin (Selection 2) and incubated at 24 ± 1 °C in the dark for 2 weeks (Fig. 1h). The number of explants per plate were reduced by approximately half at Selection 2. After 2 weeks the calli were transferred to a lit culture room under fluorescent lights (100 μmol m−2 s−1) at 24 ± 1 °C with a 16-h photoperiod and covered with a single layer of paper towel for a further week. During this period putative transformed lines should start to green and produce small shoots (Fig. 1i).
Regeneration of transgenic plants
After the 3 weeks on Selection 2 medium, the calli were transfer one final time to wheat regeneration medium (WRM) containing 4.4 g L−1 MS (Duchefa M0222), 20 mg L−1 sucrose, 0.5 mg L−1 MES supplemented with 0.5 mg L−1 Zeatin, 160 mg L−1 Timentin and 20 mg L−1 Hygromycin, 3 g L−1 Gelzan (Sigma-Aldrich) in deep Petri dishes (tissue culture dish, 90 mm diameter × 20 mm, Falcon 353003). All regenerating callus derived from a single embryo was labelled to track its origin. The paper covering was removed and the calli were cultured under fluorescent lights (100 μmol m−2 s−1) at 24 ± 1 °C with a 16-h photoperiod.
Rooting
Regenerated shoots which were 1–2 cm in length with visible roots (Fig. 1j) were transferred to “De Wit” culture tubes (Duchefa, W1607) containing 8 mL of WCI without growth regulators, solidified with 3 g L−1 Gelzan and supplemented with 160 mg L−1 Timentin and 15 mg L−1 Hygromycin. A strong root system with root hairs developed on putative transformed plants (Fig. 1k).
Acclimatisation
Regenerated plantlets with strong root systems (Fig. 1l) were gently removed from the tubes using long forceps and the roots gently washed with cool running water to remove any remaining tissue culture medium. They were planted in a peat and sand mix in 5 cm square cell trays and covered with a clear plastic propagator lid. To maintain high humidity around the plants, they remained covered with the propagator lids for approximately 1 week while they became established in soil. Within a controlled environment room, the plants were grown at 18 ± 1 °C during the day (16 h) and 15 ± 1 °C at night temperatures, with relative humidity maintained at 65%, metal halide lamps (HQI) supplemented with tungsten bulbs provided a light intensity of with 400–600 μmol m−2 s−1 a 16 h photoperiod.
GUS histochemical assay
The GUS activity was determined after co-cultivation, resting, Selection 1, after rooting medium and on T1 seed in the next generation using a GUS histochemical assay. The plants were immersed in GUS assay substrate containing 1 mmol L−1 of 5-bromo-4-chloro-3-indolyl glucuronide (X-gluc), 100 mmol L−1 sodium phosphate, 10 mmol L−1 Na2EDTA and 0.1% of triton X-100, pH = 7 at 37 °C under dark conditions overnight (~ 16 h). All green samples were decoloured and fixed in 70% ethanol to remove chlorophyll and other plant pigments prior to visualising and photographing.
DNA extraction
0.5 to 0.7 cm leaf samples were harvested in PCR tubes, and DNA was extracted by Extract-N-Amp™ Plant Tissue PCR Kits (Cat No. XNAP-1KT) following the manufacturer’s instructions.
HygR (hpt) polymerase chain reaction (PCR)
A 335 bp amplicon of the hygromycin hpt gene was PCR amplified using the primer pair HygF 5′-AGGCTCTCGATGAGCTGATGCTTT-3′, Hyg Reverse 5′-AGCTGCATCATCGAAATTGCCGTC-3′ and REDExtract-N-Amp PCR Reaction Mix (Cat No. XNAS) with a 20 µL total volume per reaction. Each reaction comprised of 10 µL PCR Reaction Mix (REDExtract-N-Amp), ~ 50 ng of plant genomic DNA, 1 µL (10 mM) of each primers (Hyg F and Hyg R), and sterile laboratory grade water up to a total volume of 20 µL. PCR was performed in a Peltier Thermal Cycler 200 (MJ Research), with the conditions 95 °C for 3 min, followed by 34 cycles of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, then 72 °C for 7 min before a final hold of 10 °C. PCR products were resolved by gel electrophoresis on a 1% agarose gel which contained ethidium bromide at 1 μg 10 mL−1.
Quantitative real-time PCR to determine transgene copy number
Approximately 100 mg leaf samples were placed into 1.5 mL Eppendorf tubes and using liquid nitrogen flash frozen. The leaf material was stored at − 80 °C if DNA extraction could not be performed immediately. DNA was extracted from the leaf material using the Qiagen DNeasy plant mini kit (Cat No. 69106) according to the manufacturer’s instructions. A Nanodrop ND-1000 spectrophotometer was used to assess DNA concentrations.
iDna Genetics performed Quantitative real-time PCR using the hygromycin resistance gene (hpt) and CO2 (Constans-like, AF490469) gene specific probes and primers as described in Bartlett et al. [2]. Using the design module “TaqMan Probe and Primer” of the Applied Biosystems software Primer Express, target sequence specific primers were designed. The reactions used low rox version of the Absolute mix (Catalogue AB1318B, ThermoScientific). Multiplex assays were performed on the hpt gene and the CO2 gene. The final concentrations of probes and primers were at 200 nM. Each assay contained 5 μL of DNA solution, which was optimised for final DNA concentrations 1.25 to 10 ng μL−1 (6.25 to 50 ng DNA in each assay). PCRs were performed in an Applied Biosystems Quantstudio5 Machine equipped with a 384-place plate. The PCR cycling conditions were 95 °C 15 min (activation of enzyme), 40 cycles of 95 °C 15 s, 60 °C 60 s.