Donor plants of the spring barley, Golden Promise, were grown under controlled environment conditions with 15°C day and 12°C night temperatures as previously described 20. Humidity was 80% and light levels 500 μmol.m^-2.s^-1 at the mature plant canopy level provided by metal halide lamps (HQI) supplemented with tungsten bulbs. Immature barley spikes were collected when the immature embryos were 1.5–2 mm in diameter. Immature seeds were removed from the spikes and sterilised as previously described 20. The immature embryos were exposed using fine forceps and the embryonic axis removed. The embryos were then plated scutellum side up on callus induction (CI) medium containing 4.3 g l^-1 Murashige & Skoog plant salt base (Duchefa), 30 g l^-1 Maltose, 1.0 g l^-1 Casein hydrolysate, 350 mg l^-1 Myo-inositol, 690 mg l^-1 Proline, 1.0 mg l^-1 Thiamine HCl, 2.5 mg l^-1 Dicamba (Sigma-Aldrich) and 3.5 g l^-1 Phytagel, with 25 embryos in each 9 cm Petri dish. 1.25 mg l^-1 CuSO₄.5H₂O was added where experiments included additional copper in the callus induction phase. Additional copper was present in the CI media of all six transformation experiments described. All media components used throughout the tissue culture process, with the exception of phytagel, were made as a double-concentrate and filter-sterilised. Phytagel was made up as a double concentrate (in H₂O) and autoclaved. After autoclaving in H₂O the phytagel could be stored at room temperature until required, and then heated to 60°C prior to mixing with the filter-sterilised media components.

Agrobacterium strain AGL1 containing one of three pBract vectors (pBract215, pBract216 or pBract217) was used in all experiments. The vectors pBract215, pBract216 and pBract217 all derive from the Gateway destination vector pBract203, which features a T-strand region containing the hpt gene (conferring hygromycin resistance) under the control of a CaMV 35s promoter and a negative selection gene (ccdB) flanked by Gateway recombination sites to assist the cloning of genes of interest (Figure 1). In pBract215, pBract216 and pBract217, the ccdB gene has been replaced by the firefly luciferase gene (luc) driven by the maize Ubi1 promoter. These vectors are all the same apart from the intron composition of their luciferase genes. pBract216 and pBract217 contain the maize RpoT intron 4 and Arabidopsis UBQ10 intron 1 respectively within the coding sequence of the luciferase gene at position +165. The pBract vectors are detailed on the bract website 21. All pBract vectors are based on pGreen 22 and therefore need to be co-transformed into Agrobacterium with the helper plasmid pSoup. To enable the small size of pGreen, the pSa origin of replication required for replication in Agrobacterium, is separated into its' two distinct functions. The replication origin (ori) is present on pGreen, and the trans-acting replicase gene (RepA) is present on pSoup. Both vectors are required in Agrobacterium for pGreen to replicate. Maps of pBract203, pBract215 and pSoup are shown in Figure 1.

The pBract vectors were independently electroporated into Agrobacterium AGL1 cells along with pSoup. Each electroporation was set up by adding 1 μl of pBract construct DNA (100 ng/μl) and 1 μl of pSoup DNA (100 ng/μl) to 40 μl of electrocompetent AGL1 cells on ice. The cells and DNA were gently mixed, and then transferred to a pre-chilled cuvette (with 2 mm separation). Electroporation was carried out using a Biorad GenePulser, with the settings: 2.50 kv, 25 μFD, 400 Ohms. The time constant was between 8.9 and 9.2 ms. The cells were recovered by adding 100 μl LB media (10 g l^-1 tryptone, 5 g l^-1 yeast extract and 10 g l^-1 NaCl) and then transferring the contents of the cuvette to 2 ml of LB. This was gently shaken at room temperature (approximately 24°C) for 2 hours. One hundred microlitres of the electroporation was plated on LB solidified with 1% agar and supplemented with 50 μg ml^-1 kanamycin and 50 μg ml^-1rifampicin. These plates were incubated at 28°C for 48 hours.

The method of Tingay et al. 2 was used to prepare a standard Agrobacterium inoculum for transformation. A 400 μl aliquot of standard inoculum was removed from -80°C storage, added to 10 ml of MG/L 23 medium without antibiotics and incubated on a shaker at 180 rpm at 28°C overnight. This full strength culture was used to inoculate the prepared immature embryos. A small drop of Agrobacterium suspension was added to each of the immature embryos on a plate. The plate was then tilted to allow any excess Agrobacterium suspension to run off. Immature embryos were then gently dragged across the surface of the medium (to remove excess Agrobacterium) before being transferred to a fresh CI plate, scutellum side down. Embryos were co-cultivated for 3 days at 23–24°C in the dark.

After co-cultivation, embryos were transferred to fresh CI plates containing 50 mg l^-1 hygromycin and 160 mg l^-1 Timentin (Duchefa). Our standard Agrobacterium-mediated transformation protocol 5 involved culture for four to six weeks on CI medium followed by two weeks on a transition medium with increased levels of copper similar to that described by Cho et al. 15, before transfer to regeneration medium. The main difference between our protocol and that of Cho et al. 15 was that our transition medium was based on the regeneration medium, not on the callus induction medium. In the improved protocol described here, copper at 1.25 mg l^-1 was added to all CI medium following the co-cultivation step. Embryos were sub-cultured onto fresh CI selection plates every 2 weeks and kept in the dark at 24°C. After 4–6 weeks, embryos were transferred to transition medium containing 2.7 g l^-1 Murashige & Skoog modified plant salt base (without NH₄NO₃) (Duchefa), 20 g l^-1 Maltose, 165 mg l^-1 NH₄NO₃, 750 mg l^-1 Glutamine, 100 mg l^-1 Myo-inositol, 0.4 mg l^-1 Thiamine HCl, 1.25 mg l^-1 CuSO₄.5H₂O, 2.5 mg l^-1 2, 4-Dichlorophenoxy acetic acid (2,4-D) (Duchefa), 0.1 mg l^-1 6-Benzylaminopurine (BAP), 3.5 g l^-1 Phytagel, 50 mg l^-1Hygromycin and 160 mg l^-1 Timentin in low light. Low light levels (75 μmol.m^-2.s^-1) were achieved by placing the plates under light conditions in a tissue culture room but covering the plates with a thin sheet of paper. After a further 2 weeks, embryo derived callus was transferred to regeneration medium in full light (140 μmol.m^-2.s^-1) at 24°C, keeping all callus from a single embryo together. Regeneration medium was the same as the transition medium but without additional copper, 2,4-D or BAP. Once regenerated plants had shoots of 2–3 cm in length they were transferred to glass culture tubes containing CI medium, without dicamba or any other growth regulators but still containing 50 mg l^-1 hygromycin and 160 mg l^-1 Timentin. Transformed plants developed a strong root system in the hygromycin containing medium in 1–2 weeks and were then transferred to soil and grown under the same conditions as the donor plants.

Luciferase assays were performed using 2 cm leaf samples taken from regenerated plantlets growing in culture tubes containing CI medium with selection. Luciferase activity was quantified according to the method of Harwood et al. 14.

PCR analysis was carried out to confirm the presence of the hpt gene for lines deriving from transformation with the constructs pBract216, pBract215 and pBract217 which were shown to have no luciferase expression. DNA was extracted (by the John Innes Genome Laboratory) from 50 mg of leaf tissue using the Qiagen DNeAsy 96 Plant kit (Q69181) system following the manufacturer's instructions. A 917-bp fragment of the hpt gene was amplified using the aphIV primers described by Vain et al. 24. Reactions were composed of 17 μl ABgene 1.1× ReddyMix™ PCR Master Mix (1.5 mM MgCl₂), 0.5 μM of each primer and 20 – 35 ng template DNA. PCR was carried out using a Peltier Thermal cycler PTC-200 (MJ Research), with the conditions 95°C for 1 minute, 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 1 minute, return to step 2 30 times, 72°C for 10 minutes. PCR products were separated on 1% agarose gels (containing 10 μg of ethidium bromide per 100 ml) using gel electrophoresis.

Differences in transformation frequencies were assessed using chi-square analysis. Two-way tables giving the numbers of transformed/untransformed embryos for each variable were entered into Genstat (version 9.1.0.147). For each 2-way table, Genstat computed a Pearson chi-square value, and from this generated the p-value. Significance was assessed at the 5% level.

DNA was extracted by the same method used for the hpt PCR (described above). Quantitative real-time PCR was carried out by iDna Genetics using luciferase and CO2 (Constans-like, AF490469) gene specific primers and probes. Primers were designed to the target sequences using Applied Biosystems software Primer Express, with the TaqMan Probe and Primer design module (Table 1). The reactions used ABGene Absolute QPCR Rox Mix (Cat number AB1139). The luciferase gene and the CO2 gene were assayed in multiplex. The probes and primer final concentrations were 200 nM. The assay contained 5 μl of DNA solution, and was optimised for final DNA concentrations 1.25 to 10 ng/μl (6.25 to 50 ng DNA in the assay). PCRs were carried out in an Applied Biosystems ABI7900 equipped with a 384 place plate. The detectors used were FAM-TAMRA and VIC-TAMRA with Rox internal passive reference, and no 9600 emulation. The PCR cycling conditions were 50°C 2 minutes hold, 95°C 10 minutes (enzyme activation), 40 cycles of 95°C 15 seconds, 60°C 60 seconds.

Eight lines transformed with pBract216 were subject to Southern analysis. For each line, fifteen micrograms of genomic DNA were digested with 40 units of BamHI (which cuts once within hpt and once between ubi1 and luc) and separated on a 0.8% agarose gel. An alkaline Southern blot was assembled using a Hybond-N+ membrane (Amersham) according to manufacturer's instructions. DNA was fixed to the membrane by cross-linking in a UV Stratalinker 2400 (Stratagene). Probe DNA was produced by PCR using the primers LUCF (5'-GCCGGTGTTGGGCGCGTT-3') and LUCR (5'-GCGGGAAGTTCACCGGCG-3') to amplify a 1176 bp fragment of the luc coding sequence. The probe was labelled with ³²P α-dCTP prior to hybridisation. The blot was imaged using a Typhoon 9200 Phosphorimager (Amersham).

Development of the high-throughput Agrobacterium-mediated barley transformation system was dependent on the availability of immature embryos from good quality plants grown under controlled conditions. All immature embryos used in the present study were from plants of the spring genotype Golden Promise, grown under controlled conditions. Plants were not sprayed with fungicides or insecticides at any stage of growth. Our earlier work indicated that poor transformation results could often be linked to poor quality donor material or to occasions when plants had been sprayed (unpublished). In addition, particular care was taken with the watering regime so that the plants were never too dry or overwatered. Starting with good quality immature embryos, an experiment was initiated to look at the effect of additional copper in the culture medium on regeneration efficiency.

Our standard protocol was compared to a method that included additional copper (5 μM CuSO₄) in the CI medium as well as in the transition medium. This study was carried out without any Agrobacterium treatment to examine regeneration ability from immature embryos only. Fifty-eight immature embryos with the embryonic axis removed were used in this study and were split between two different culture regimes. Thirty embryos were cultured on callus induction medium (CI) (standard protocol) and twenty-eight embryos were cultured on callus induction medium supplemented with 5 μM CuSO₄ (CI + Cu). The appearance of the embryo derived callus after 27 days of culture is shown in Figure 2. There was more callus growth on the CI medium with copper (Figure 2b) than on the standard CI medium (Figure 2a). In addition, detailed examination of the callus revealed that as well as showing faster growth, the callus on the copper containing medium appeared more embryogenic. After four weeks on callus induction medium, all callus lines were transferred to transition medium for two weeks and then to regeneration medium. All callus derived from each embryo was kept separate and after 4 weeks on regeneration medium the shoots derived from each embryo were removed and counted.

Figure 3 shows the average number of shoots regenerated per embryo with the two different tissue culture regimes. The standard protocol, gave an average of 26 shoots per embryo. The improved protocol, containing copper in the CI medium, gave an average of 53 shoots per embryo. Adding the additional copper during callus induction therefore led to approximately double the number of regenerated shoots per embryo up to the point when the shoots were scored. The shoots derived from embryos cultured on CI + Cu were also larger than those cultured without copper. It appears that the regeneration process was accelerated by the presence of copper in the CI medium as embryogenic callus formed earlier leading to the more rapid regeneration of plants.

These two protocols were compared in some small preliminary transformation experiments (data not shown). The number of transformed callus lines obtained was unaffected by the additional copper, however the number of transformed plants obtained as a proportion of the number of starting embryos was significantly higher when copper was added to the callus induction medium. Improved plant regeneration therefore appeared to increase transformation efficiency. This initial finding was further examined in large scale experiments described below.

Six transformation experiments (A – F, detailed in Table 2) were carried out to test the improved barley transformation protocol, using a total of 1750 immature embryos. These experiments utilised the three constructs, pBract215, 216 and 217, which are identical apart from the intron content of their luciferase gene. For these experiments, the improved regeneration regime (with 5 μM CuSO₄ in CI media) was used. In addition, all culture media were filter sterilised and embryos were collected from healthy, well maintained plants that had not been sprayed at any stage of growth. These were the key differences from our previous protocol 5. Some previous studies have recommended using freshly isolated embryos whereas others have included pre-culture of embryos prior to inoculation. In the present experiments, the effect of modifying the timing of embryo isolation/inoculation upon transformation efficiency was investigated by inoculating embryos on both the same day as isolation and the day after isolation in four of the six experiments.

Experiments A, B, C and F (Table 2) were each performed using embryos inoculated on both the same day and the day after isolation. These experiments showed no significant effect of embryo isolation/inoculation timing on transformation efficiency (p = 0.342) (Table 3). Combining the results of all six experiments, 150 independent transformed lines were created from 575 embryos inoculated on the day after isolation (giving a transformation efficiency of 26.1%) and 282 independent transformed lines were produced from 1175 embryos inoculated on the same day as isolation (giving a transformation efficiency of 24.0%).

The improved method was applied to a total of 1750 immature embryos, and of these, 432 were successfully transformed into independent hygromycin-resistant plants, giving an average transformation efficiency of 24.7% (Table 2). Plants producing strong roots in hygromycin containing medium were found to be transformed in 98.5% of cases. There was no significant difference in transformation efficiency between constructs (p = 0.062) and between experiments (p = 0.098) (Table 3). The transformed status of 333 independent lines was checked by analysis of luciferase expression (data not shown). Of these lines, 95.5% were expressing the luciferase gene. Two thirds of the 4.5% of lines not expressing luciferase were subsequently shown (by PCR) to contain the hygromycin resistance gene (hpt) – validating their transformed status. The remaining lines (1.5% of the total analysed for luciferase expression) did not contain the hpt gene and were therefore considered untransformed. These 5 lines were omitted from the transformation efficiency calculations (Tables 2 and 3) and their possible origin is discussed later. Plants from 328 of the independent lines were grown to maturity and of these 321 produced seed giving a 98% fertility rate.

Luciferase copy number was determined for 260 independent hygromycin-resistant lines using real-time PCR. The distribution of copy numbers obtained is given in Figure 4. Of the transformed plant lines analysed, 8 (3%) did not contain an intact copy of the luciferase gene. These lines were not escapes, as they were shown by PCR to contain the hpt gene. Forty-six percent of the transformed lines contained a single copy of the luciferase gene, and only 8% of the lines contained more than three copies. The distribution of copy numbers obtained was consistent across the three constructs used. Southern analysis was performed to check the luc copy number and integration pattern in eight T₀ lines transformed with pBract216 (Figure 5). For seven of the lines, the number of T-DNA insertion sites identified by Southern analysis was less than or equal to the number of luc copies predicted by qPCR, as would be expected (Table 4).