Hayta et al. Plant Methods (2019) 15:121 https://doi.org/10.1186/s13007-019-0503-z Plant Methods

METHODOLOGY Open Access An efcient and reproducible Agrobacterium‑ mediated transformation method for hexaploid wheat (Triticum aestivum L.) Sadiye Hayta*, Mark A. Smedley, Selcen U. Demir, Robert Blundell, Alison Hinchlife, Nicola Atkinson and Wendy A. Harwood

Abstract Background: Despite wheat being a worldwide staple, it is still considered the most difcult to transform out of the main cereal crops. Therefore, for the wheat research community, a freely available and efective wheat transformation system is still greatly needed. Results: We have developed and optimised a reproducible Agrobacterium-mediated transformation system for the spring wheat cv ‘Fielder’ that yields transformation efciencies of up to 25%. We report on some of the important factors that infuence transformation efciencies. In particular, these include donor plant health, stage of the donor material, pre-treatment by centrifugation, vector type and selection cassette. Transgene copy number data for inde- pendent plants regenerated from the same original immature embryo suggests that multiple transgenic events arise from single immature embryos, therefore, actual efciencies might be even higher than those reported. Conclusion: We reported here a high-throughput, highly efcient and repeatable transformation system for wheat and this system has been used successfully to introduce genes of interest, for RNAi, over-expression and for CRISPR– Cas9 based genome editing. Keywords: Wheat, Triticum aestivum, Genetic modifcation, Agrobacterium tumefaciens, Immature embryo

Background using particle bombardment (biolistics) of embryogenic Hexaploid wheat (Triticum aestivum L.) is one of the callus tissue [7]. Te frst reported Agrobacterium- “big three” cereal crops after maize (Zea mays) and rice mediated transformation of wheat was to follow in 1997 (Oryza sativa) [1–3]. It is unrivalled in its geographic [8]. Nevertheless, in spite of this frst promising report, range of cultivation and accounts for approximately 20% biolistic-mediated transformation of wheat remained the calorifc value and 25% of daily protein intake of the method of choice for some considerable time, as wheat world’s population [1]. Wheat has arguably, more infu- transformation via Agrobacterium continued to be chal- ence on global food security than any other crop [4]. In lenging and inefcient [9]. Reports in the literature of spite of wheats global importance, it has lagged behind Agrobacterium-mediated wheat transformation generally other main cereals, primarily rice and maize, in the devel- describe low transformation efciencies of around 5%. opment of genomic tools for its improvement and is still Efcient wheat transformation via an in planta Agrobac- considered the most challenging of the major cereals to terium-mediated inoculation method was reported by transform [5, 6]. Risacher et al. [10] however, this methodology required In the early 1990’s the frst report was published of specialist skills and has not been widely adopted. transgenic wheat being produced by direct DNA transfer Another efcient patented transformation system is available through licence from Japan Tobacco Inc (http:// *Correspondence: [email protected] www.jti.co.jp), licenced as two systems, the basic PureIn- Department of Crop Genetics, John Innes Centre, Norwich Research Park, tro™ and the more advanced PureUpgrade™. Ishida et al. Norwich, Norfolk NR4 7UH, UK

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Hayta et al. Plant Methods (2019) 15:121 Page 2 of 15

[11], described the process, and reported efciencies of was taken to restrict unnecessary staf access to the con- 40–90% however, our lab and others (personnel com- trolled environment rooms, hairnets and designated lab munication) have not been able to replicate the process coats were maintained in a freezer (− 20 °C) to reduce the based on the published information. It appears that the risk of spreading pathogens. specialist training provided to laboratories licensing the technology is a prerequisite to successful reproduction Immature embryos isolation of the method and/or specialist vectors are required. Wheat spikes were collected approximately 14 days post Terefore, a robust, reproducible and transferrable wheat anthesis (dpa), when the immature embryos (IE) were transformation system that is widely available to the 1–1.5 mm in diameter (Fig. 1c, d) and early milk stage research community is still needed. GS73 [16]. Kernels from foret 1 and 2 on central spikelet For any transformation method the procedure can be (Fig. 1a, b) were used for transformation. Te awns were easily split into two component phases: those that enable cut of the ears approximately 3–5 mm from the grain. efcient T-DNA transfer and incorporation into the plant Te seed coat can be removed but this was not essen- genome and those that allow the selection of transformed tial unless contamination problems are encountered. cells and regeneration of whole transgenic plants [12]. In Te immature grains were separated from the ear and wheat, some factors afecting T-DNA transfer include; the placed in a 150 mL Sterilin jar. Within a laminar airfow binary vector used in conjunction with Agrobacterium cabinet under aseptic conditions, the grains were surface strain, the inclusion of additional vir genes, pre-treatments sterilised using 70% ethanol (v/v) for 1 min, given 1 rinse of embryos, Agrobacterium inoculation and co-cultivation. with sterile distilled water, followed by 7 min in 10% (v/v) Te key factors infuencing wheat regeneration in vitro sodium hypochlorite (Fluka 71696). Te grains were then include; the cultivar used, quality and health of donor washed 3 times with sterile distilled water. material, stage of immature embryos, handling of material, All subsequent operations were performed under ster- and media composition including all components from ile conditions in a laminar fow hood. Embryos were iso- nutrients to gelling agents and phytohormones [9, 13, 14]. lated from the immature grains using fne forceps under a In this present study we report a reproducible Agrobac- dissecting microscope. Approximately 100 embryos were terium-mediated transformation system for the spring put into two 1.7 mL Eppendorf tubes (Fig. 1e) containing wheat Fielder that yields transformation efciencies 1 mL wheat inoculation medium (WIM) modifed from up to 25%. Tis system has been widely used to intro- [11] containing 0.44 mg L−1 Murashige and Skoog (MS) duce genes of interest as well as for CRISPR/Cas9 based [17] plant salt base (Duchefa M0222), 10 g L−1 glucose, genome editing [15]. 0.5 g L−1 2-(N-morpholino) ethanesulfonic acid (MES), 0.05% Silwet L-77 and 100 µM Acetosyringone (AS) Materials and methods added fresh just before use. Plant material Seeds of the spring wheat (Triticum aestivum L.) cv Construct assembly ‘Fielder’ were sown at weekly intervals in a mixture of To create a pGreen [18] based, Golden Gate cloneable peat and sand (85% fne grade peat, 15% washed grit, vector compatible with the Modular Cloning (MoClo) 4 kg m−3 maglime, 2.7 kg m−3 Osmocote (3–4 months), system a 799 bp fragment containing the LB and a LacZ −3 1 kg m PG Mix 14-16-18 + Te 0.02% and wetting agent). Golden Gate cassette was isolated from pAGM8031 Tey were initially sown in 5 cm diameter pots and after (Addgene 48037) using restriction enzymes PshAI/PmeI, approximately 4 weeks the germinated plants were trans- and cloned into pGreen II 0000 at the HpaI/StuI sites ferred into 13 cm diameter pots containing John Innes using blunt end ligation. Tis Level 2 binary vector was Cereal Mixture (40% medium grade peat, 40% sterilised deemed pGoldenGreenGate-M (pGGG-M) (Fig. 2a). loam (soil), 20% washed horticultural grit, 3 kg m−3 A reporter pGGG vector was made, for wheat trans- −3 maglime, 1.3 kg m PG mix 14-16-18 + Te base fertiliser, formation, which contained the hygromycin resist- −3 1 kg m Osmocote mini 16-8-11 2 mg + Te 0.02%, and ance gene (Hpt) and Cat1 intron driven by the rice wetting agent) for continued development. Plants were actin1 promoter, and the β-glucuronidase gene with 2 grown in controlled growth chambers (Conviron Europe introns (GUS2Int) driven by the rice ubiquitin promoter Ltd) at 20 ± 1 °C day and 15 ± 1 °C night temperatures, (Fig. 2b). Briefy, the Level 1 constructs pICH47802- 70% humidity with light levels of 800 μmol m−2 s−1 pro- RActpro::HptInt::NosT (selectable maker) and vided by fuorescent tubes and tungsten lighting. At any pICH47742-RUbipro::GUS2int::NosT (GUS Reporter) stage of growth, the donor plants were not sprayed with were cloned into the binary Level 2 vector pGGG-M insecticides or fungicides. In addition, particular care using standard Golden Gate MoClo assembly [19]. Hayta et al. Plant Methods (2019) 15:121 Page 3 of 15

Fig. 1 a–c Selection of wheat spikes and immature embryos at the correct stage, d isolated immature embryo, e immature embryos in Eppendorf tube containing 1 mL WIM, f immature embryos on co-cultivation medium, g immature embryo with the embryonic axis removed before transferring to resting medium, h callus induction on Selection 1 and Selection 2 media, i transformed callus starting to green and produce small shoots, j regenerated shoots with visibly strong roots, k transgenic wheat plant transferred to culture tube showing strong root system in hygromycin containing medium, l transgenic wheat plants before transferring to soil

a pSa-ORI b Ubi 3 intron (rice)

LB Ubiquitin 3 Promoter (rice) Gus exon1 2nd LB Act1 promoter (Oryza sativa) intron1 npt1 Bbs I (748) CAT1 intron (Ricinus communis) Gus exon2 P lac prom pGoldenGreenGate (pGGG) P(LAC) HygR (hpt) CDS intron 2 3377 bp HindIII (943) nos terminator Gus exon3 SphI (953) 2nd LB nos terminator PstI (959)

SalI (961) LB RB

XbaI (967)

Sma I (980)

KpnI (986) colEI ori SacI (992) EcoRI (994) ALPHA Bbs I (1345) pGGG Wheat GUS marker RB Fig. 2 a The pGoldenGreenGate-M (pGGG-M) a GoldenGate (MoClo) level 2 vector based on pGreen. b pGGG containing the rice actin promoter driving the hygromycin (hpt) selection gene containing the CAT1 intron and the rice ubiquitin promoter driving the GUS maker gene containing two introns

Preparation of Agrobacterium for transformation and incubated at 28 °C, shaken at 200 rpm for ~ 65 h. A Te hypervirulent Agrobacterium tumefaciens strain modifed method of Tingay et al. [23] to prepare Agro- AGL1 [20] was used in all plant transformation experi- bacterium standard inoculums for transformation was ments. Vectors were electroporated into Agrobacterium used as previously described by Bartlett et al. [2]. Equal AGL1 competent cells as previously described [2], when quantities of 30% sterile glycerol and the Agrobacterium pGreenII [18] derivatives were used i.e. pBRACT [21] or culture were mixed by inverting and aliquots of 400 μL pGGG they were co-electroporated with the helper plas- in 0.5 mL Eppendorf tubes were made. Te aliquots of mid pSoup [18] or its derivatives pAL154 contained the standard inoculums were frozen at − 80 °C and stored 15 kb Komari fragment or pAL155 with an additional until required. VirG gene. Te day before wheat transformation a single 400 μL Single colonies of Agrobacterium AGL1, which con- standard inoculum was used to inoculate 10 mL of liquid tain the desired vector, were inoculated into 10 mL of LB MG/L [24] (5 g L−1 mannitol, 5 g L−1 tryptone, 2.5 g L−1 [22] liquid medium containing appropriate antibiotics yeast, 100 mg L−1 NaCl, 1 g L−1 Glutamic acid, 250 mg L−1 Hayta et al. Plant Methods (2019) 15:121 Page 4 of 15

−1 −1 Tis transfer is referred to as Selection 1. Te calli were ­KH2PO4, 100 mg L MgSO­ 4, 1 μg L Biotin. fnal pH = 7) medium without antibiotics and incubated at 28 °C shaken split at the next transfer into clumps of approximately 2 at 200 rpm overnight (~ 16 h). On the day of transforma- 4 mm− , callus pieces derived from each single embryo tion, the bacteria were pelleted by centrifugation in a were labelled to keep track of their origin. Te calli were 50 mL Falcon tube at 3100 rpm for 10 min at 24 °C. Te transferred to fresh selection plates (WCI) as above, 1 supernatant was discarded, and the cells resuspended gen- but with 30 mg L− Hygromycin (Selection 2) and incu- tly in 10 mL wheat inoculation medium (WIM) to an opti- bated at 24 ± 1 °C in the dark for 2 weeks (Fig. 1h). Te cal density of 0.5 OD (600 nm) and 100 µM AS added. Te number of explants per plate were reduced by approxi- culture was incubated at room temperature with gentle mately half at Selection 2. After 2 weeks the calli were agitation (80 rpm) for 4–6 h in the dark. transferred to a lit culture room under fuorescent lights −2 −1 (100 μmol m s ) at 24 ± 1 °C with a 16-h photoperiod Inoculation with Agrobacterium and co‑cultivation and covered with a single layer of paper towel for a fur- Te isolated embryos were placed into fresh WIM ther week. During this period putative transformed lines medium prior to centrifugation at 14,000 rpm at 4 °C should start to green and produce small shoots (Fig. 1i). for 10 min [25]. WIM was removed with a pipette and after 1 mL Agrobacterium solution added, the tubes Regeneration of transgenic plants were inverted frequently for 30 s and incubated at room After the 3 weeks on Selection 2 medium, the calli were temperature for at least 20 min. After the incubation transfer one fnal time to wheat regeneration medium 1 period, the Agrobacterium suspension was poured with (WRM) containing 4.4 g L− MS (Duchefa M0222), 1 1 the embryos into a 50-mm diameter Petri plate and the 20 mg L− sucrose, 0.5 mg L− MES supplemented 1 1 Agrobacterium suspension was removed with a pipette. with 0.5 mg L− Zeatin, 160 mg L− Timentin and 1 1 Te embryos were transferred, scutellum side up, to the 20 mg L− Hygromycin, 3 g L− Gelzan (Sigma-Aldrich) co-cultivation medium which consisted of WIM sup- in deep Petri dishes (tissue culture dish, 90 mm diam- −1 plemented with 100 μM AS, 5 µM ­AgNO3, 1.25 mg L eter × 20 mm, Falcon 353003). All regenerating callus −1 derived from a single embryo was labelled to track its ori- CuSO­ 4·5H2O and 8 g L agarose [11]. Twenty-fve embryos were placed in each 90 mm single vent Petri gin. Te paper covering was removed and the calli were 2 1 plate (Termo Scientifc No 101R20) and incubated at cultured under fuorescent lights (100 μmol m− s− ) at 24 1 °C with a 16-h photoperiod. 24 ± 1 °C in the dark for 3 days co-cultivation (Fig. 1f). ± Troughout the tissue culture process, all solid media components, except for the gelling agent, were prepared Rooting as a double-concentrate and flter-sterilised. Te gelling Regenerated shoots which were 1–2 cm in length with agents were prepared as a double concentrate in water visible roots (Fig. 1j) were transferred to “De Wit” culture and sterilised by autoclave. After autoclaving the gel- tubes (Duchefa, W1607) containing 8 mL of WCI with- out growth regulators, solidifed with 3 g L−1 Gelzan and ling agents (2×) were maintained at 60 °C and the flter- supplemented with 160 mg L−1 Timentin and 15 mg L−1 sterilised media components (2×) were warmed to 60 °C prior to mixing both and pouring. Te phytohormones Hygromycin. A strong root system with root hairs devel- and antibiotics were added as flter sterilised stocks just oped on putative transformed plants (Fig. 1k). before pouring. Acclimatisation Resting period, callus induction and selection Regenerated plantlets with strong root systems (Fig. 1l) of transformed material were gently removed from the tubes using long forceps and After 3 days’ co-cultivation, the embryogenic axes were the roots gently washed with cool running water to remove excised from the embryos using forceps (Fig. 1g). Te any remaining tissue culture medium. Tey were planted embryos were transferred to the fresh callus induc- in a peat and sand mix in 5 cm square cell trays and cov- tion plates (WCI) based on the media described in ered with a clear plastic propagator lid. To maintain high [26] but containing 2 mg L−1 Picloram (Sigma-P5575), humidity around the plants, they remained covered with 0.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D), the propagator lids for approximately 1 week while they 160 mg L−1 Timentin and 5 mg L−1 agarose and incu- became established in soil. Within a controlled environ- ment room, the plants were grown at 18 1 °C during the bated at 24 ± 1 °C in the dark for 5 days. Timentin was ± added to control Agrobacterium during the resting day (16 h) and 15 ± 1 °C at night temperatures, with rela- period. Te embryos were transferred, scutellum side up, tive humidity maintained at 65%, metal halide lamps (HQI) to fresh WCI plates as above with 15 mg mL−1 Hygro- supplemented with tungsten bulbs provided a light inten- sity of with 400–600 μmol m−2 s−1 a 16 h photoperiod. mycin and incubated at 24 ± 1 °C in the dark for 2 weeks. Hayta et al. Plant Methods (2019) 15:121 Page 5 of 15

GUS histochemical assay specifc primers were designed. Te reactions used low Te GUS activity was determined after co-cultivation, rox version of the Absolute mix (Catalogue AB1318B, resting, Selection 1, after rooting medium and on T1 TermoScientifc). Multiplex assays were performed seed in the next generation using a GUS histochemical on the hpt gene and the CO2 gene. Te fnal concentra- assay. Te plants were immersed in GUS assay substrate tions of probes and primers were at 200 nM. Each assay containing 1 mmol L−1 of 5-bromo-4-chloro-3-indolyl contained 5 μL of DNA solution, which was optimised glucuronide (X-gluc), 100 mmol L−1 sodium phosphate, for fnal DNA concentrations 1.25 to 10 ng μL−1 (6.25 −1 10 mmol L Na­ 2EDTA and 0.1% of triton X-100, pH = 7 to 50 ng DNA in each assay). PCRs were performed in at 37 °C under dark conditions overnight (~ 16 h). All an Applied Biosystems Quantstudio5 Machine equipped green samples were decoloured and fxed in 70% ethanol with a 384-place plate. Te PCR cycling conditions were to remove chlorophyll and other plant pigments prior to 95 °C 15 min (activation of enzyme), 40 cycles of 95 °C visualising and photographing. 15 s, 60 °C 60 s.

DNA extraction 0.5 to 0.7 cm leaf samples were harvested in PCR tubes, Results and DNA was extracted by Extract-N-Amp™ Plant Tissue Optimization of wheat transformation with Agrobacterium PCR Kits (Cat No. XNAP-1KT) following the manufac- Efect of gelling agent on callus induction and regeneration turer’s instructions. An initial study was performed to examine the regen- eration capacity of wheat IEs, without an Agrobacterium HygR (hpt) polymerase chain reaction (PCR) treatment, 40 embryos were cultured per gelling agent A 335 bp amplicon of the hygromycin hpt gene was PCR with four diferent gelling agents (2% Gelzan G1910, 5% amplifed using the primer pair HygF 5′-AGGCTC​ TCG​ ​ Agarose A9045, 3.5% Phytagel P8169 Sigma-Aldrich, ATGAGC​ TGA​ TGC​ TTT​ -3​ ′, Hyg Reverse 5′-AGC​TGC​ and 8% Agar AGA03 For Medium) to identify which gel- ATCATC​ GAA​ ATT​ GCC​ GTC​ -3​ ′ and REDExtract-N- ling agent yielded the highest percentage of regenerated Amp PCR Reaction Mix (Cat No. XNAS) with a 20 µL plants. total volume per reaction. Each reaction comprised of Troughout the 5-week callus induction phase of 10 µL PCR Reaction Mix (REDExtract-N-Amp), ~ 50 ng this experiment there was little diference visible to the of plant genomic DNA, 1 µL (10 mM) of each primers naked eye in callus development on each type of gelling (Hyg F and Hyg R), and sterile laboratory grade water up agent. However, when examined under the microscope, to a total volume of 20 µL. PCR was performed in a Pel- embryo-derived callus on Agarose or Gelzan appeared to tier Termal Cycler 200 (MJ Research), with the condi- have a better embryogenic structure (Fig. 3a). tions 95 °C for 3 min, followed by 34 cycles of 95 °C for Te calli were transferred to WRM with the four dif- 30 s, 58 °C for 30 s, 72 °C for 1 min, then 72 °C for 7 min ferent gelling agents. A notable diference in the perfor- before a fnal hold of 10 °C. PCR products were resolved mance of the gelling agents was observed after 4 weeks by gel electrophoresis on a 1% agarose gel which con- on WRM. Te majority of shoots that regenerated on tained ethidium bromide at 1 μg 10 mL−1. Agar and Phytagel remained stunted for the remainder of regeneration stage; whilst the shoots on Gelzan, and Quantitative real‑time PCR to determine transgene copy Agarose had extensive shoot development reaching in number excess of 10 cm in length (Fig. 3b). Te gelling agents per- Approximately 100 mg leaf samples were placed into formance in terms of the percentage of IE-derived calli to 1.5 mL Eppendorf tubes and using liquid nitrogen fash produce shoots larger than 1 cm was Gelzan 81%, Agrose 77.5%, Phytagel 51.5% and Agar 57.5%. For all further frozen. Te leaf material was stored at − 80 °C if DNA extraction could not be performed immediately. DNA experiments we chose to use Agarose for callus induction was extracted from the leaf material using the Qiagen and Gelzan for regeneration and rooting. DNeasy plant mini kit (Cat No. 69106) according to the Pre‑treatment and co‑cultivation manufacturer’s instructions. A Nanodrop ND-1000 spec- trophotometer was used to assess DNA concentrations. To examine the efect of the centrifugation pre-treat- iDna Genetics performed Quantitative real-time PCR ment, 150 IE were isolated and placed in Eppendorf tubes using the hygromycin resistance gene (hpt) and CO2 containing WIM, half the embryos (75) were subjected (Constans-like, AF490469) gene specifc probes and to centrifugation at 14,000 rpm at 4 °C for 10 min. Te primers as described in Bartlett et al. [2]. Using the design controls without centrifugation were incubated at 4 °C module “TaqMan Probe and Primer” of the Applied for 10 min. Both centrifuged and control treatments Biosystems software Primer Express, target sequence had the WIM removed and were then inoculated with Hayta et al. Plant Methods (2019) 15:121 Page 6 of 15

a Gelzan Agarose Phytagel Agar

2 weeks

4 weeks

b

2 weeks

3 weeks

5 weeks

Fig. 3 a The efect of gelling agent on callus formation on WCI with Gelzan, Agarose, Phytagel and Agar, maintained in the dark. b The efect of gelling agent on shoot regeneration from wheat calli when cultured on WRM with Gelzan, Agarose, Phytagel and Agar, maintained under 16 h light

Agrobacterium AGL1 containing pGGG as previously while in the control group, only 50% of embryos and 50% described in the methods section. Te IE’s were then of calli were GUS positive. Te degree of the blue foci co-cultivated for 3 days. Subsets of 10 embryos/embryo after resting and frst selection was stronger in the centri- derived calli were sacrifced for GUS staining at two fuged group than the untreated control. Between groups stages; after the resting period and after the frst selection the diference was statistically signifcant (P < 0.05) after stage. In the centrifuged group, 90% of the embryos and the resting period. Te remaining calli, 55 in each treat- 60% of the calli chosen for GUS staining showed blue foci, ment, were taken through the entire transformation Hayta et al. Plant Methods (2019) 15:121 Page 7 of 15

process. Te number of individual embryos yielding calli Length of co‑cultivation that regenerated shoots was higher in the pre-treated Te efect of co-cultivation period (2 days or 3 days) centrifuged group when compared to the untreated con- was investigated; embryos were isolated and inoculated trol group. Plants were regenerated from 15 calli out of 55 on the same day, then transferred to resting medium (27.3%) that were derived from the embryos pre-treated either after 2 days or 3 days co-cultivation. GUS stain- by centrifugation, while only three embryos derived calli ing was performed after the resting period and after the regenerated plants (5.5%) from the control group. Ten of frst selection. Twenty-fve embryos/calli per group were the regenerated plants expressed GUS in the centrifuged checked for their GUS expression (Fig. 4b). Although group giving a transformation efciency of 18.2%. Of the 100% of the embryos/calli stained blue in both groups, untreated control group only one plant expressed GUS, embryos co-cultured for 3 days had stronger GUS stain- giving 1.8% transformation efciency. Terefore, centrif- ing than those co-cultured for 2 days. Using the same ugation at 14,000 rpm at 4 °C for 10 min was used in our scoring system, the diference between groups was statis- all transformation experiments. tically signifcant (P < 0.05) after resting. We used 3 days co-cultivation time in all further experiments. Efect of embryo orientation during culture Immature embryos were cultured with their scutellum Transformation efciency, selection and plasmid backbone side either in contact with the medium or facing upwards Te improved protocol, as reported earlier in the meth- during and after their co-cultivation with Agrobacterium ods section, was applied to 3289 IEs over 27 experiments, AGL1 containing pGGG. Te diferences between the which gave rise to 380 transformed plants, our transfor- two groups in terms of GUS staining were tested after mation efciencies ranged from 5 to 25% (Table 1). Sig- co-cultivation, a resting period and on the frst selec- nifcant diferences in transformation efciencies were tion medium. One hundred embryos were used as start- found between the three constructs using pGGG (18%), ing material in each group and 25 embryos/calli per pAGM (12%) and pBRACT (5%) (P < 0.05) (Fig. 5). Fur- group were tested for GUS expression after each stage. thermore, signifcant diferences in transformation ef- Te staining was scored under a light microscope. Each ciencies were seen when diferent promoters were used individual embryo/callus was given a score. Te increas- (CaMV 35S or rice actin) to drive the hygromycin (Hpt) ing number of the score was an indicator of strength resistance gene containing the CAT1 intron (P < 0.001). (degree) of the staining. Scoring started from 0 (no GUS Actin:hpt in either pGGG or pAGM backbones outper- expression seen), score 1 (1–5 foci ~ quarter of the mate- formed the 35S:hpt selection in pBRACT (Fig. 5). Te rial), score 2 (5–10 foci ~ half the material) score 3 (10– plasmid backbone containing the actin:hpt selection was 15 ~ three quarters of the material) and score 4 (almost found to have a signifcant efect on transformation ef- totally stained). Te statistical analyses showing the ciency (P < 0.001). Te pGGG vector gave a higher aver- transformation efciency diferences between the groups age transformation efciency (18.22%) when compared were performed using unpaired t test in Genstat 18th to pAGM8031 (12.03%), both constructs contained edition software. A P-value less than 0.05 was considered an identical actin:hpt selection cassette. Lines created to be statistically signifcant. with the pBRACT construct, containing the 35s:hpt, Based on transient expression of the GUS gene, gave the greatest number of escapes (non-transformed embryos co-cultured scutellum side up had more stain- lines) coming through the transformation process, out ing than those cultured scutellum side down. Although of 93 regenerated plantlets, 11 were escapes (11.8%), 100% of the embryos tested were stained blue in both when compared to actin:hpt selection at 133 plantlets, 8 groups, scutellum side up co-cultured embryos showed escapes (6%). To help eliminated escapes, it was observed blue foci on both embryo sides, whereas embryos co- that if plants produced strong roots which showed root cultured scutellum side down had mainly GUS staining hairs in media containing hygromycin selection, they on the axis side periphery. Similarly, after the resting were usually found to be transformed, whereas, those period and frst selection, calli from embryos cultured without root hairs were escapes, later confrmed by PCR scutellum side up had more blue foci than those cultured and/or GUS staining (Fig. 6a). Figure 6b shows gus gene scutellum side down and GUS staining was stronger expression in T1 seeds showing segregation in the next (Fig. 4a). Te diference between groups was signif- generation. cant (P < 0.05) after cultivation on Selection 1 medium according to the scoring system of GUS stained embryos mentioned above. IEs were cultured scutellum side up Copy number results throughout the transformation procedure in all subse- Te copy number data revealed diferences between each quent experiments. of the constructs used. Te highest percentage of single Hayta et al. Plant Methods (2019) 15:121 Page 8 of 15

Fig. 4 a GUS staining images of scutellum down (the frst column) and up (the second column) groups after co-cultivation/resting/selection 1. b GUS staining images of co-cultivation 2 days (the frst column) and 3 days (the second column) groups after co-cultivation/resting/selection 1

T-DNA insertion lines were created using the pBRACT events and that the occurrence of multiple independent construct, with 38.5% of lines being single copy, pGGG events from single embryos is common. gave rise to 29.1% single copy lines. Te pAGM construct Initial studies were performed to assess the efect, on gave the lowest percentage of single wheat copy lines at transformation efciency, of including additional viru- 15.8% (Fig. 7). Wheat lines created using the pAGM con- lence (vir) genes on pSOUP derivatives within Agro- struct gave rise to more two copy lines (22.6%) than lines bacterium AGL1. Te GUS reporter pGGG construct with a single copy, furthermore, 27.8% of lines created was included in AGL1 along with either the standard using this construct had high copy number, containing pSOUP plasmid, or its derivatives pAL154 contained more than 10 transgene copies (Fig. 7). Te pGGG and the 15.2 kb Komari fragment, or pAL155 with an addi- pBRACT constructs produced 19% and 38.5% respec- tional virG542 gene. Isolated IEs were treated as previ- tively, of high copy lines with more than 10 copies of the ously described in the method section and sacrifced transgene (Fig. 7). for GUS staining 5 days after Agrobacterium inocula- Transgene copy number analysis was performed on tion. Twenty-four embryos were used per treatment multiple individual plants arising from the same single and experiments repeated twice. Diferences were not embryos in a subset consisting of 24 embryonic lines. seen between the embryos treated with AGL1 contain- Te copy number difered between plants taken from ing pGGG plus the standard pSOUP and AGL1 con- the same embryo in 19 of the 24 lines tested (79.2%). taining pGGG plus pAL155 virG542, 94.4% and 95.83% Tis suggests that these are independent transformation respectively. However, embryos treated with AGL1 Hayta et al. Plant Methods (2019) 15:121 Page 9 of 15

Table 1 Efects of promoter driving hygromycin selection and plasmid backbone on transformation efciency BackBone Promoter driving the selectable Number of inoculated IE Number of independent Transformation marker gene transgenic plants efciency (%) pGGG​ Act Hyg Intron 150 14 9 pGGG​ Act Hyg Intron 175 27 15 pGGG​ Act Hyg Intron 160 40 25 pGGG​ Act Hyg Intron 100 18 18 pGGG​ Act Hyg Intron 25 5 20 pGGG​ Act Hyg Intron 25 6 24 pGGG​ Act Hyg Intron 80 19 24 pGGG​ Act Hyg Intron 175 12 7 pAGM Act Hyg Intron 100 15 15 pAGM Act Hyg Intron 100 12 12 pAGM Act Hyg Intron 100 5 5 pAGM Act Hyg Intron 100 14 14 pAGM Act Hyg Intron 100 24 24 pAGM Act Hyg Intron 200 14 7 pAGM Act Hyg Intron 125 21 17 pAGM Act Hyg Intron 150 13 9 pAGM Act Hyg Intron 150 16 11 pAGM Act Hyg Intron 80 18 23 pAGM Act Hyg Intron 150 11 7 pAGM Act Hyg Intron 125 11 9 pAGM Act Hyg Intron 150 15 10 pAGM Act Hyg Intron 150 10 7 pAGM Act Hyg Intron 100 11 11 pBract 35S Hyg Intron 80 4 5 pBract 35S Hyg Intron 150 10 7 pBract 35S Hyg Intron 100 5 5 pBract 35S Hyg Intron 100 5 5 pBract 35S Hyg Intron 100 5 5 pBract 35S-Hyg-Intron 39 2 5 pBract 35S-Hyg-Intron 50 3 6

20.0 18.0 ) 16.0 14.0 12.0 lines (% 10.0 8.0 6.0

Transgenic 4.0 2.0 0.0 Act Hyg Intron Act Hyg intron 35S Hyg Intron pGGG pAGM pBract Axis Title Fig. 5 Transformation efciencies as percentages for the three constructs used in this study pGGG, pAGM and pBRACT​ Hayta et al. Plant Methods (2019) 15:121 Page 10 of 15

Fig. 6 a Transgenic plants showing GUS expression after being on rooting medium and Fielder (non-transformed) tissue culture control on the

right. b Segregating ­T1 generation seeds showing GUS expression in three independent transgenic lines

45.0 40.0

) 35.0 30.0

lines (% 25.0 20.0 15.0

Transgenic 10.0 5.0 0.0 123456789>10 Copy number

pAGM-ActHyg Intron pGGG-Act Hyg Intron pBract 35S-Hyg Intron Fig. 7 Comparison of copy number data using the diferent plasmid backbones and selection cassettes containing pGGG plus pAL154 showed GUS staining in cut in half and stained for GUS as described in “Materi- 54.16% of embryos. als and methods” section. Non-transformed Fielder seed were used as a negative control. Two of the lines tested Transgene inheritance and segregation each had 33 seeds which were GUS positive (blue) and All transgenic plants produce in this study had a nor- 15 GUS negative (white) null segregants, the remain- mal phenotype and set seed. To prove germline inherit- ing line had 37 GUS positive seed and 11 white negative ance of the GUS transgene GUS analysis was performed null segregants. From the 144 seeds tested, if following on dried mature T1 seed. Tree single copy transgenic a Mendelian inheritance ratio of 3:1, one would expect T1 lines (copy number determined by qPCR) were cho- 108 GUS positive and 36 GUS negative null segregants. sen. 48 seed from each T1 line were randomly selected, Te observed 103 GUS positive and 41 GUS negative null Hayta et al. Plant Methods (2019) 15:121 Page 11 of 15

segregants is not signifcantly diferent to the expected 2 Ear collecon and surface (X P = 0.505602 not signifcant at P < 0.05), and therefore follows a 3:1 inheritance ratio. sterilisaon

Discussion Plant transformation technologies which enable genetic Isolaon of immature embryos modifcation are invaluable tools for functional genomic studies and crop improvement programmes [21]. Trans- formation efciency for wheat has languished around Centrifugaon 5% for many years despite its global importance [14]. In the present study we have developed an efcient, repro- ducible and transferrable transformation method for Inoculaon of embryos with wheat. Te transformation process takes approximately Agrobacterium 11 weeks (Fig. 8) and has been used successfully over sev- eral years. We report some of the key factors infuencing wheat transformation efciency and provide a detailed Co-culvaon 3 days reproducible protocol. Conveniently, stable plant transformation can be divided into two components: the phases that enable Resng 5 days DNA transfer to the cell and integration into the genome efectively, and those which enable the selection and regeneration from transformed cells of whole viable Selecon 1 2 weeks plants [27]. Te driving force behind improvements to transformation systems are the increased demands for Selecon 2 systems that are high-throughput, highly efcient and 3 weeks cost-efective [9]. Tese improvements are partly due to the development of improved tools for gene delivery and Regeneraon 2-3 weeks genetic manipulation and through tissue culture regime refnement enabling better plant regeneration. DNA cloning approaches and the technologies used to imple- Roong 2 weeks ment them, underpin and are often the starting point for most gene function studies [21]. In our experience, a key feature essential for success is Soil good quality, heathy, donor material grown under con- Fig. 8 Timeline showing the main steps of Agrobacterium-mediated trolled environmental conditions. Donor plants should transformation in wheat not be sprayed with pesticide at any growth stage, there- fore, good plant hygiene practices need to be in place. Te conditions of the controlled growth chamber should be adjusted to produce vigorous growing plant material improved DNA delivery and transformation efciency. so that the IEs reach the early milk stage GS73, approxi- However, the mechanism by which the centrifugation mately, 14 days post anthesis. Collection of IEs that are pre-treatment afects transformation is still unknown. healthy and at the correct stage is key. We examined whether the orientation of the immature wheat embryos during culture afected expression of the Pre‑treatment and co‑cultivation GUS reporter gene. Traditionally, wheat embryos are cul- Pre-treatment by centrifugation, embryo orientation tured scutellum upward, as are maize embryos, within during co-cultivation with Agrobacterium and the dura- the transformation process, whereas, barley embryos tion of co-cultivation were all important factors afecting are cultured scutellum down, with the scutella in con- DNA delivery and transformation efciency in wheat. tact with the medium surface [29]. In the frst reported Silwet L-77 usage and centrifuge pre-treatment were Agrobacterium-mediated transformation of wheat, by already proven to increase the wheat transformation ef- Cheng et al. [8], embryos were cultured scutellum up. ciency of IEs [8, 11, 28]. Pre-treatment by centrifugation We found that embryos cultured scutellum side up gave is assumed to increase the cell wall membrane perme- stronger GUS staining after the resting period and the ability and the penetrance of Agrobacterium, resulting in frst selection. Hayta et al. Plant Methods (2019) 15:121 Page 12 of 15

Te majority of wheat transformation protocols ambig- Te pBRACT vector, also used in this study, contains uously state that co-cultivation of embryos with Agro- the 35S promoter driving an intron enhanced hygromy- bacterium is performed for 2 to 3 days [8, 14, 30–32]. cin resistance gene and is highly efcient at transforming Although, some are specifc, 2 days [11, 33] or 3 days [34]. barley [2, 21]. Te CaMV 35S promoter does express in In this study, under the reported conditions, we found monocotyledonous plants, but its comparative strength is that 3 days co-cultivation had signifcantly stronger GUS considerably lower in monocotyledonous than in dicoty- expression when tested after the resting period, how- ledonous plant cells [37]. Transgene expression in trans- ever, after the frst selection the diference was not as genic plants can be strongly enhanced by the inclusion of pronounced. Tis might suggest an increased in T-DNA introns in many cases [38]. Te inclusion of a castor bean delivery and transient gene expression, but DNA integra- catalase-1 (CAT-1) intron within the Hpt gene has been tion and stable gene expression may be similar in both shown to increase transgene expression by approximately treatments. 2.5 fold and improved transformation efciency in rice and barley [39, 40]. Te use of monocot derived promot- Gelling agent ers to drive transgenes within monocot transgenic plants Te main constraint for enhancing Agrobacterium-medi- has resulted in a high degree of gene expression [37]. Our ated wheat transformation is the capacity to efectively results concur with Jang et al. [37], in that, in wheat, the regenerate transgenic plants from transformed callus. We 35S promoter within pBRACT was successively out per- investigated the efect of diferent gelling agents (Gelzan, formed by the monocot derived rice actin (OsAct1) pro- Agarose, Phytagel and Agar) on the regeneration from moter when driving the intron enhanced Hpt gene within IEs of the wheat cultivar Fielder over a 9-week culture pGGG or pAGM. period. Te purpose was to identify which gelling agent yielded the highest quantity of shoots larger than 1 cm Agrobacterium tumefaciens strains and binary vectors in length and the highest proportion of regenerating An Agrobacterium strains ability to transform plant cells embryos. is determined by its plasmid and chromosomal genomes, In terms of regeneration frequency throughout the two all the machinery necessary for cell attachment and experiments, Gelzan (81%) and Agarose (77.5%) were the DNA-transfer is encoded by them [14]. Tere are only most consistent at producing shoots > 1 cm and produced two chromosomal backgrounds that have been used the highest average number of shoots. Phytagel and Agar successfully to transform wheat, one is Ach5 of strain were consistently poor regarding regeneration frequency. LBA4404 [41] the other is the C58 background, both Te infuence of fve diferent gelling agents has also have been used with a variety of Ti plasmids and binary been reported by Berrios et al. [35], looking at Phytagar, vectors. An important group of C58 strains that have Phytagel, Agarose, Arcagel and Agar–Agar. Tey used played key roles in wheat transformation are EHA101, multiple recombinant inbred lines of sunfower cotyle- EHA105, AGL0, and AGL1 [20, 42, 43], originating from dons. Teir fndings similarly reported signifcant dif- strain A281 that contains the hypervirulent pTiBo542 Ti ferences depending on the gelling agent used. Agarose plasmid which harbours additional vir genes. Te intro- induced positive regeneration by organogenesis. duction to Agrobacterium of vir gene copies or combina- tions, has been achieved by using alternative Ti plasmids, Efect of selection cassette and plasmid backbone additional helper plasmids or on the backbone of binary In the present study, the frst requirement was a binary vectors containing additional virulence genes [44]. Te vector that allowed efcient and straightforward wheat ability of Agrobacterium strains containing the hyperviru- transformation. We required the ease and speed of the lent Ti plasmid pTiBo542 to confer higher transformation modular cloning (MoClo) system based on type IIS efciencies in wheat have been demonstrated in several restriction enzyme cloning “Golden Gate assembly” comparative studies [32, 45]. Te present study utilises as described by [19]. Furthermore, we required an easy the hypervirulent Agrobacterium strain AGL1 containing method for including additional virulence genes within pTiBo542. Transformation of wheat with Agrobacterium the strain of Agrobacterium as several previous publi- LBA4404, a weakly virulent strain, was only successful cations report the importance of additional virulence when a superbinary vector was used that contained addi- genes within wheat transformation experiments [14, 36]. tional vir B, C and G genes from pTiBo542 [46]. Te ben- Hence, we developed the MoClo compatible pGolden- efcial efect of extra vir genes was illustrated by increased GreenGate (pGGG) vector based on pGreen and there- T-DNA transmission and transformation of wheat when fore, compatible with the helper plasmid pSoup and its the 15 Kb Komari fragment from pTiBo542 was included derivatives containing additional virulence genes [18]. on the pSoup helper plasmid in the hypervirulent strain Hayta et al. Plant Methods (2019) 15:121 Page 13 of 15

AGL1 [47]. Wang et al. [48] found that transforma- interactions at many levels occur during the transfor- tion efciencies doubled, to 4%, with the addition of the mation process, interactions between the plant mate- Komari fragment or an additional vir gene regulator virG. rial, bacterial cell, bacterial chromosome, Ti plasmid and In our initial transient expression studies, the inclusion of binary vector. the Komari fragment (virB/C/G) on the pSoup derivative We have demonstrated that the GUS gene is stably pAL154 was detrimental to T-DNA transfer, yielding 54% transmitted to the next generation and is expressed in ­T1 GUS stained embryos compared to the control pSoup or seeds (Fig. 6b). A Mendelian inheritance ratio of 3:1 was the pSoup derivative pAL155 with the additional virG542, observed in transgene transmission, as one would expect both yielding ~ 95% of embryos displaying GUS staining. from single copy T-DNA transgenic lines. Te addition of pAL154 (virB/C/G) with pGGG within Te transformation efciency throughout the paper the Agrobacterium may have caused an imbalance in was calculated as single events occurring from each the transformation machinery. In stable transformation embryogenic line (1 plant from 1 embryo) and displayed experiments, that compared the standard pSoup plasmid as the percentage of positive transgenic plants produced and pAL155 virG542, a slight, non-statistically signif- from the total number of IEs isolated and inoculated with cant, improvement was seen in transformation efciency Agrobacterium in an experiment. However, copy number 542 when the additional virG was used, 17 ± 3.2% com- data showed that plants derived from a single embry- pared to 19 ± 4%. Te high ~ 95% of embryos showing onic line had diferent copy numbers, 19 of the 24 lines GUS expression in our transient studies would suggest tested (79.2%). Tis suggests that multiple transformation that future further improvement in transformation ef- events, from single embryogenic lines, occur often and ciencies will be achieved through improvement in plant our transformation efciency might be much higher than regeneration rather than with improved DNA transfer. we have calculated. Te incorporation of additional vir genes into binary vectors is not always necessary, a substantial amount of transgenic lines using normal Agrobacterium strains and Conclusion binary vectors have been documented [14]. Developing enhanced functional wheat genomics tools is In experiments comparing the vector backbones, we of the utmost importance for the wheat scientifc com- found that pGGG had a higher transformation ef- munity and breeders. Here we report a high-throughput, ciency than pAGM, 18% compared to 12% respectively. highly efcient and repeatable transformation protocol We also found that pGGG gave a higher percentage of for the wheat cv ‘Fielder’. Te protocol has been suc- transgenic lines containing single copy T-DNAs com- cessfully transferred to other research groups. Work is pared to pAGM, 29.1% compared to 15.8%. In bacteria, underway to further test this protocol in other wheat the plasmid origin of replication determines the number cultivars. New technologies such as genome editing, of plasmid copies within a bacterial cell. Binary plasmids using CRISPR–Cas systems rely heavily on efcient, contain two origins of replication, one enables replication reproducible transformation systems. Our method has within E. coli, the second, “broad-spectrum” origin of already been used extensively for both gene characterisa- replication, allows replication within Agrobacterium. Te tion studies and genome editing in wheat. It is extremely broad-spectrum replication origin contained on pGGG probable that even greater efciencies will be accom- is pSa ori and on pAGM it is pVS1 ori, plasmids with plished with further optimisation, allowing more rapid these origins are maintained at around 4 copies per cell exploitation of new genomic resources and genome edit- and 7–10 copies per cell, respectively [49, 50]. Te addi- ing technologies. tional copies of pAGM within Agrobacterium, in com- Acknowledgements parison to pGGG, may ofer some insight into explaining We are grateful to all the BRACT group for their support. Thank you to Mark the higher T-DNA copies found integrating in transgenic Youles of TSL SynBio for supplying Golden Gate components. Also, we thank lines transformed with pAGM. A restricted number of JIC Horticulture and photographic services. T-strands transmitted to the plant cell may lead to low Authors’ contributions incorporation and therefore lower transgene copy num- SH developed the method, carried out data assessment and drafted the paper. MAS performed molecular cloning, contributed to the analysis, participated bers in plants. A correlation between plant and bacterial to draft the paper. SUD and RB contributed the initial experiments. AH and T-DNA copy number, may be expected, when transform- NA participated to optimise the transformation system. WAH supervised and ing with diferent plasmids containing diferent origins coordinated the project and edited the manuscript. All authors read and of replication, as they replicate to varying extents within approved the fnal manuscript. the Agrobacteria [50]. T-DNA copy number may also Funding be infuenced by the strain of Agrobacterium, method We acknowledge funding from BBSRC through Grant ‘BBS/E/J/000PR9778’ of transformation and target tissue used [51]. Multiple along with any other acknowledgements. Hayta et al. Plant Methods (2019) 15:121 Page 14 of 15

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