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. 2020 Jun 25;37(2):177–184. doi: 10.5511/plantbiotechnology.19.1216d

Low-temperature pretreatment of explants and high maltose concentration during callus culture improves particle-bombardment-mediated stable transgene expression in common wheat

Fumitaka Abe 1,*, Shingo Nakamura 1, Masahiko Mori 1,a, Ikuo Ashikawa 1
PMCID: PMC7434674  PMID: 32821225

Abstract

Biolistic transformation systems are widely used to introduce foreign genes into common wheat (Triticum aestivum L.); however, these techniques often generate high transgene copy numbers and complex transgene integration patterns that hinder the stable expression of the transgenes. To improve the efficiency of stable transgene expression, we examined the effect of low-temperature pretreatment of wheat flower spikes and of high maltose concentration (HMC) in the medium during the subsequent callus culture. Tillers of the spring wheat cultivar Bobwhite were stored at 5°C without water for one week before the isolation of their immature scutellar tissues, and the resulting particle-bombarded explants were cultured on 15% maltose for a month. Together, these treatments significantly increased the number of recovered transgenic lines expressing the reporter gene. The low-temperature pretreatment eliminated the negative effects of HMC, and HMC improved the efficiency of stable transgene expression. Southern blot analysis revealed that transgenic lines recovered after HMC treatment integrated a lower copy number of transgenes than those cultured at normal (4%) maltose concentration. These findings suggest that the HMC-mediated reduction of the transgene copy number results from the suppression of plasmid DNA rearrangement before or during transgene integration into the wheat genome.

Keywords: biolistic, copy-number, high maltose concentration, low-temperature pretreatment, Triticum aestivum

Introduction

Wheat (Triticum aestivum) is a major crop around the world and a primary target for the improvement of agronomic traits using genetic engineering technology. Because of its large and complex genome, it can be difficult to translate findings from the study of model plants such as rice (Oryza sativa) directly into wheat research. The establishment of high-throughput transformation systems in wheat is therefore important for its genetic analysis and genetic engineering. Over the past few decades, numerous teams have studied wheat tissue culture and transformation (see reviews by Barcelo et al. 2001; Janakiraman et al. 2002; Jones 2005; Sahrawat et al. 2003); however, despite the importance of wheat transformation techniques, it remains one of the most difficult crops to transform.

Among the several approaches used for wheat transformation, the most widely used is still the particle bombardment, or biolistic transformation, of tissues derived from immature embryos (Harwood 2012). Biolistic transformation often results in the integration of multiple transgenes with extremely complex rearrangements, which hinders the stable expression of the transgenes (Barcelo et al. 2001; Kohli et al. 2003). The use of linear transgene cassettes rather than circular plasmids in biolistic transformation has been proposed as a way of regulating transgene integration patterns in rice (Breitler et al. 2002; Fu et al. 2000) and wheat (Ismagul et al. 2018; Tassy et al. 2014; Yao et al. 2006); however, the physiological state of the cell is likely to regulate integration more than any feature of the transgene itself (Breitler et al. 2002; Kohli et al. 2003). The state of the cells in the target explants and cultured tissues can be altered by the environmental conditions in which the donor plant was grown or the composition of the callus culture medium subsequently used, among other factors. The environmental conditions experienced by the donor plant influence the efficiency of somatic embryogenesis and shoot regeneration from immature wheat embryos (Hess and Carman 1998; Pastori et al. 2001).

Several attempts have been made to prepare wheat explants for tissue culture and transformation. Among them, stress pretreatment of explants has been suggested to improve the efficiency of somatic embryogenesis, and is therefore considered useful in wheat biolistic transformation (Li et al. 2003a, 2003b; Pellegrineschi et al. 2002a). Furthermore, during biolistic transformation, osmotic treatments can limit cell damage by causing plasmolysis, thereby minimizing cytoplasmic leakage from the cells after bombardment (Vain et al. 1993). Short-term osmotic treatments spanning a few hours before and after bombardment have been successfully used in cereals (Altpeter et al. 1996; Brettschneider et al. 1997; Chen et al. 1998; Harwood et al. 2000; Rasco-Gaunt et al. 1999). The use of a relatively high sucrose concentration (9%) throughout the callus culture period improves regeneration efficiency and subsequent transformation efficiency in wheat (Rasco-Gaunt et al. 2001); thus, a high osmotic condition affects the efficiency of wheat biolistic transformation.

In this study, we tested modifications of the explant and callus culture conditions that alter the cell states in target explants and cultured tissues to examine their impact on the efficiency of stable transgene expression in wheat biolistic transformation. The combination of the low-temperature pretreatment (LTP) of explants and the culture of calli under a high maltose concentration (HMC) significantly improved the efficiency of the stable transgene expression. Analyses of the transgene copy number and inheritance patterns suggest that HMC suppressed the rearrangement of transgenes.

Materials and methods

Plant materials, growth conditions, and explant pretreatments

The spring wheat variety ‘MPB Bobwhite 26’ (Bobwhite) (Pellegrineschi et al. 2002b) was used in all experiments. Four seeds were sown into each 18-cm pot containing a 2 : 1 mixture of two composts, Sakata Soil Mix (Sakata Seed, Japan) and Kureha fertilized granulated soils (Kureha, Japan). The plants were grown in a glasshouse at approximately 15°C with an 8 h light/16 h dark photoperiod. After 12 weeks, the plants were transferred to a controlled environmental chamber with adjusted day/night temperatures of 16°C/10°C under a 14 h light/10 h dark photoperiod.

The LTP of the explants was performed as follows. Tillers (13–15 days post anthesis) were harvested by detaching them from the main plant below their third node, retaining the three leaves, and were placed in a refrigerator at 5°C without water for 5–7 days. Non-pretreated control explants were prepared from spikes at 17–19 days post anthesis. The immature embryos in the pretreated and non-pretreated spikes were developmentally equivalent, with both categorized as the middle of developmental stage II described by Rogers and Quatrano (1983).

Isolation of scutellar tissues from immature embryos

Immature caryopses were collected from the pretreated or control spikelets, rinsed with 70% ethanol, sterilized with a solution of sodium hypochlorite, 0.5% (v/v) available chlorine, containing 0.1% (v/v) Tween 20 for 15 min, and then rinsed three times with sterile distilled water. The immature embryos were isolated aseptically under a dissecting microscope, and the entire embryonic axis was removed using a fine blade. The isolated scutellar tissues were cultured, scutellum side up, on callus induction medium containing 0.2 M mannitol (CI-0.2Man, Table 1) at 25°C in the dark for 3–4 h before bombardment. A total of 32 scutellar tissues were cultured per 90-mm plate, and all scutellar tissues were numbered so that they could be traced.

Table 1. Composition of media.

Medium name Composition
Callus induction-0.2 M mannitol (CI-0.2 Man) MS salts and vitamins, 0.2 M mannitol, 1 mg l−1 thiamin-HCl, 1.95 g l−1 MES, 3.5 mM Gln, 1 mM Pro, 1 mM Asn, 100 mg l−1 casein hydrolysate (pH 5.8), 2 g l−1 phytagel (Sigma), autoclaved. Picloram (2 mg l−1), 2 mg l−1 2,4-dichlorophenocyacetic acid (2,4-D), 100 mg l−1 ascorbic acid, and 150 g l−1 maltose were added after autoclaving.
Callus induction-5 mg l−1 Bialaphos (CI-5B) Same as CI-0.2Man, but without mannitol. Bialaphos (5 mg l−1) was added after autoclaving.
Callus maintenance-5 mg l−1 Bialaphos (CM-5B) Same as CI-5B, but 0.5 mg l−1 2,4-D.
Shoot growth-2 mg l−1 Bialaphos (SG-2B) Macrosalts (1.4 g l−1 KNO3, 300 mg l−1 NH4NO3, 200 mg l−1 KH2PO4, 450 mg l−1 CaCl2·2H2O, 350 mg l−1 MgSO4·7H2O); microsalts (40 mg l−1 Fe·Na·EDTA, 11.2 mg l−1 MnSO4·5H2O, 5 mg l−1 H3BO3, 7.5 mg l−1 ZnSO4·7H2O, 0.75 mg l−1 KI, 0.25 mg l−1 Na2MoO4·7H2O, 0.025 mg l−1 CuSO4·5H2O, 0.025 mg l−1 CoCl2·6H2O); 30 g l−1 maltose, 200 mg l−1 myo-inositol (pH 5.7), 5 g l−1 agar, autoclaved. Zeatin (5 mg l−1), 0.1 mg l−1 2,4-D, vitamins (10 mg l−1 thiamin-HCl, 1 mg l−1 pyridoxine-HCl, 1 mg l−1 nicotinic acid, 1 mg l−1 Ca-pantothenate, 1 mg l−1 ascorbic acid), and 2 mg l−1 bialaphos were added after autoclaving.
Root growth-5 mg l−1 Bialaphos (RG-5B) Same as SG-2B, but without zeatin and 2,4-D, and with 5 mg l−1 bialaphos.
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Particle bombardment

The scutellar tissues were bombarded with the plasmid pAL156 (Figure 1), which contains the bar selectable marker gene and the modified β-glucuronidase (uidA) reporter gene, both under the control of the maize Ubiquitin1 (ubi-1) promoter and first intron (Christensen and Quail 1996). Plasmid DNA was prepared using a Qiagen Maxi Kit (Qiagen). A 5-µg aliquot of the plasmid DNA was precipitated in a mixture consisting of 2 mg gold particles (1.0 µm in diameter), 1 M CaCl2, and 16 mM spermidine. Gold particles coated with the plasmid were washed twice with ethanol and resuspend in 100 µl ethanol. For each bombardment, 5 µl of the gold particle suspension was used (100 µg particles per shot). The particle bombardments were carried out using a PDS 1000/He (Bio-Rad Laboratories) with a target distance of 5.5 cm from the stopping screen using a helium pressure of 900 psi.

Figure 1. Schematic diagram of the transformation vector, pAL156. The vector contains the bar gene and a modified uidA gene with an intron within the open reading frame (In-GUS). Both genes are driven by the maize Ubiquitin1 promoter plus the Ubiquitin1 intron (PUbi1+In). Tnos: terminator of nopaline synthase. The region used as a probe is indicated by a horizontal bar. The positions of the restriction sites (DraI) used for the Southern blot analysis and the resulting fragment size are indicated above.

Figure 1. Schematic diagram of the transformation vector, pAL156. The vector contains the bar gene and a modified uidA gene with an intron within the open reading frame (In-GUS). Both genes are driven by the maize Ubiquitin1 promoter plus the Ubiquitin1 intron (PUbi1+In). Tnos: terminator of nopaline synthase. The region used as a probe is indicated by a horizontal bar. The positions of the restriction sites (DraI) used for the Southern blot analysis and the resulting fragment size are indicated above.

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Tissue culture and selection of transgenic plants

The standard callus culture medium for scutellar tissues consisted of a modified MS (Murashige and Skoog 1962) medium (detailed composition provided in Table 1). After 16 h of bombardment, the scutellar tissues were cultured on a callus induction medium containing 5 mg l−1 bialaphos (CI-5B) at 25°C for 5 day before being transferred to a callus maintenance medium also containing 5 mg l−1 bialaphos (CM-5B). During the callus culture, 4% and 15% (w/v) maltose concentrations were used as the normal and high osmotic conditions, respectively. A total of 16 scutellar tissues were cultured per 90-mm plate. The explants were maintained intact on CM-5B for three weeks and then transferred to a shoot growth medium containing 2 mg l−1 bialaphos (SG-2B), with eight calli per plate, and cultured at 25°C under a 14 h light/10 h dark photoperiod. After three weeks, the regenerating tissues were transferred onto a root growth medium containing 5 mg l−1 bialaphos (RG-5B), and cultured under the same condition. After a further three weeks, plants showing resistance to bialaphos were selected and transferred into soil.

PCR genotyping

PCR techniques were used to screen plants to identify those harboring transgenes. Genomic DNA was isolated from leaf samples using a DNeasy Plant Mini Kit (Qiagen). The presence of bar was determined by the amplification of a 421-bp gene fragment using the primer pair 5′-GGT CTG CAC CAT CGT CAA CC-3′ and 5′-GTC ATG CCA GTT CCC GTG CT-3′. The presence of uidA was determined by the amplification of a 499-bp gene fragment using the primer pair 5′-TTG AAC TGC GTG ATG CGG AT-3′ and 5′-ACT GCC TCT TCG CTG TAC AG-3′.

GUS activity assay

The histochemical GUS assay was used to assess the expression of the transgene. Young leaf fragments were stained as described by Wu et al. (2003).

Southern blot analysis

The CTAB DNA extraction protocol (Murray and Thompson 1980) was used to isolate genomic DNA from the leaf tissues of the transgenic plants revealed to harbor transgenes in the PCR analysis. A 30-µg aliquot of DNA was digested with DraI, releasing a 4.63-kb fragment containing the uidA gene expression cassette (Figure 1). Estimates of the transgene copy number were made by comparing the intensities of the hybridizing bands from the samples and plasmid DNAs equivalent to two, four, and eight transgene copies added to non-transformed wheat DNA. The digested DNA was separated on 0.9% (w/v) agarose gels using electrophoresis and transferred onto a positively charged nylon membrane (Sigma-Aldrich). The blots were hybridized using PCR-generated probes produced using the uidA primers listed above and labeled with digoxygenin (DIG). The hybridization and chemiluminescence detection of the hybridizing bands were carried out according to the DIG Application Manual for Filter Hybridization (Sigma-Aldrich).

Statistical analysis

The use of LTP of explants and of HMC during callus culture was evaluated for its effects on the efficiencies of both transgene introduction and transgene expression. The efficiency of the transgene introduction, expressed as the number of lines containing the selectable marker gene (bar) per number of bombarded scutellar tissues ×100, was calculated on the basis of the transgenic lines confirmed using the PCR analysis. The efficiency of the transgene expression, expressed as the number of the lines showing GUS activity per number of bombarded scutellar tissues ×100, was determined using the results of the histochemical GUS assay in the T0 generation. In each procedure, the standard deviation of the efficiencies was calculated and a least significant difference pairwise comparison of the means was used to test the significance of the differences. A chi-squared (χ2) test was used to confirm the probability that the ratios conformed to the expected Mendelian segregation patterns of 3 : 1 or 15 : 1.

Results

The effect of LTP and HMC on transformation efficiency

To improve the biolistic transformation system in wheat, we examined the effects of LTP of the explants and of the use of HMC during callus culture. We prepared isolated immature scutellar tissues from plant materials following the LTP or from the non-treated controls, and then particle-bombarded the explants and cultured them on medium containing either a normal (4%) or high (15%) concentration of maltose. We assessed the efficiencies of the transgene introduction and transgene expression using a PCR analysis and a GUS histochemical assay, respectively. We conducted nine independent experiments for each procedure (designated procedures C (control), P (pretreatment; LTP), H (high-osmotic; HMC), and PH (P+H); see Figure 2). A PCR analysis of 52 transgenic lines confirmed the integration of the transgenes, showing that all of these lines contained both the bar and uidA genes. Of these transgene-containing lines, 18 showed GUS activity in the T0 generation (one representative example is shown in Figure 3A).

Figure 2. Flow diagram showing the experimental procedures. The effects of two conditions, the low-temperature pretreatment of explants and the use of a high (15%) maltose concentration during callus culture, on the efficiency of a wheat biolistic transformation were examined. The four procedures were designated as follows: procedure C (control), explants were not pretreated and were cultured on a 4% maltose medium; procedure P (pretreatment), explants were pretreated with low temperatures and cultured on a 4% maltose medium; procedure H (high-osmotic), explants were not pretreated and were cultured on a 15% maltose medium; procedure PH (pretreatment+high-osmotic), explants were pretreated and cultured on a 15% maltose medium.

Figure 2. Flow diagram showing the experimental procedures. The effects of two conditions, the low-temperature pretreatment of explants and the use of a high (15%) maltose concentration during callus culture, on the efficiency of a wheat biolistic transformation were examined. The four procedures were designated as follows: procedure C (control), explants were not pretreated and were cultured on a 4% maltose medium; procedure P (pretreatment), explants were pretreated with low temperatures and cultured on a 4% maltose medium; procedure H (high-osmotic), explants were not pretreated and were cultured on a 15% maltose medium; procedure PH (pretreatment+high-osmotic), explants were pretreated and cultured on a 15% maltose medium.

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Figure 3. Representative GUS histochemical staining, immature scutellar tissues before bombardment, and cultured calli. (A) GUS-stained young leaves of a primary transgenic plant (left) and a non-transgenic plant (right) derived from the same embryo. (B and C) Immature scutellar tissues before bombardment with (C) or without (B) the low-temperature pretreatment of explants. Arrowheads indicate the immature scutellar tissues from which the transgenic plant was obtained. (D–G) Cultured calli two weeks after bombardment using procedures C (control; D), H (high-maltose concentration; E), P (low-temperature pretreatment; F), and PH (P+H; G). Bars=2 mm.

Figure 3. Representative GUS histochemical staining, immature scutellar tissues before bombardment, and cultured calli. (A) GUS-stained young leaves of a primary transgenic plant (left) and a non-transgenic plant (right) derived from the same embryo. (B and C) Immature scutellar tissues before bombardment with (C) or without (B) the low-temperature pretreatment of explants. Arrowheads indicate the immature scutellar tissues from which the transgenic plant was obtained. (D–G) Cultured calli two weeks after bombardment using procedures C (control; D), H (high-maltose concentration; E), P (low-temperature pretreatment; F), and PH (P+H; G). Bars=2 mm.

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The efficiency of the transgene introduction for procedures C, P, H, and PH were 1.25%, 4.17%, 0.65%, and 4.17%, respectively, and the efficiency of the transgene expression for procedures C, P, H, and PH were 0.31%, 0.60%, 0%, and 2.08%, respectively (Figure 4). The efficiency of the transgene introduction was highest when the explants were pretreated with low temperatures (Figure 4; procedures P and PH). The efficiency of the transgene expression was 6.7-fold higher in the plants exposed to the PH procedure compared with procedure C (Figure 4), which was a significant increase. The efficiencies of both transgene introduction and transgene expression were lowest in plants exposed to procedure H, which did not result in any transgenic lines showing GUS activity. Typically, the size of immature scutellar tissues was smaller and thicker by the LTP of explants (representative examples shown in Figure 3B and 3C), and the increase in the callus volume over two weeks culture was smaller under HMC than under normal osmotic conditions (representative examples shown in Figure 3D–G).

Figure 4. The efficiency of the transgene integration and expression for each procedure. Nine independent experiments were conducted for each procedure, each involving more than 300 embryos. The data are represented as the mean±SE Asterisk represents a significant difference from the control (p<0.05).

Figure 4. The efficiency of the transgene integration and expression for each procedure. Nine independent experiments were conducted for each procedure, each involving more than 300 embryos. The data are represented as the mean±SE Asterisk represents a significant difference from the control (p<0.05).

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Transgene copy number

To investigate the copy number of the transgenes, we carried out a Southern blot analysis. We selected six lines, half showing GUS activity and the remainder not, from the lines transformed with procedures C, P, and PH. From procedure H, only two lines lacking GUS activity were used. The hybridization patterns were specific to each wheat line, confirming that each transgenic line originated from an independent transformation event (Figure 5). We detected the 4.63-kb uidA fragment in all lines showing GUS activity, and in six of the 11 lines not showing GUS activity. We also detected other transgene-specific bands, which indicated the presence of rearrangements in the transforming plasmid.

Figure 5. Southern blot analysis of the obtained transgenic plants. A 30-µg aliquot of genomic DNA from either transgenic or non-transformed (NT) plants was digested with DraI and blotted onto a membrane. The blot was probed with GUS (Figure 1). Plasmid DNA representing two (2C), four (4C), and eight (8C) copies of the introduced gene was mixed with non-transformed genomic DNA and included as a copy-number standard. The concentration of loaded plasmid in relation to the genome size of wheat was 16 Gbp=21 pg/2C. The size of the intact uidA gene expression cassette is indicated at right (4.63 kb). M: λHindIII. The pretreatments of the explants, maltose concentration during callus culture (see Figure 2), and comparative results of the histochemical GUS assay (see Table 2) are indicated at the top. The letter(s) in the transgenic line names represent the procedure abbreviations (see Figure 2).

Figure 5. Southern blot analysis of the obtained transgenic plants. A 30-µg aliquot of genomic DNA from either transgenic or non-transformed (NT) plants was digested with DraI and blotted onto a membrane. The blot was probed with GUS (Figure 1). Plasmid DNA representing two (2C), four (4C), and eight (8C) copies of the introduced gene was mixed with non-transformed genomic DNA and included as a copy-number standard. The concentration of loaded plasmid in relation to the genome size of wheat was 16 Gbp=21 pg/2C. The size of the intact uidA gene expression cassette is indicated at right (4.63 kb). M: λHindIII. The pretreatments of the explants, maltose concentration during callus culture (see Figure 2), and comparative results of the histochemical GUS assay (see Table 2) are indicated at the top. The letter(s) in the transgenic line names represent the procedure abbreviations (see Figure 2).

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We estimated the copy number of the uidA gene in each line by comparing the signal intensity from the transgenic lines to that of the copy number standards used. All of the transgenic lines regenerated from calli cultured in the medium containing 4% maltose (procedures C and P) contained more than nine copies of the uidA gene, whereas only a quarter of the lines derived from calli cultured in 15% maltose (procedures H and PH) contained more than nine copies (Table 2). This result indicated that the transgene copy number in the transgenic plants was suppressed when the lines were regenerated from calli cultured in HMC.

Table 2. Copy number and segregation of the transgenes.

Linesa Copy number GUS activity in T0 plants PCR positive T1 plantsb No. of locic GUS positive T1 plantsb
uidA cassette (4.63-kb) Other bands Total
Recovered from the 4% maltose condition
C1 4 5 9 — 40/48 1
C2 5 5 10 — 36/48 1
C3 8 3 11 — 26/48 na
C4 3 14 17 + 69/86 1 0/86
C5 5 7 12 + 68/84 1 49/84
C6 10 11 21 + 38/47 1 0/47
P1 2 12 14 — 10/14 1
P2 0 12 12 — sterile
P3 0 9 9 — 46/57 1
P4 7 4 11 + 57/66 2 56/66
P5 12 0 12 + 24/47 na 0/47
P6 2 7 9 + sterile
Recovered from the 15% maltose condition
H1 0 5 5 — 39/49 1
H2 0 9 9 — 57/60 2
PH1 1 4 5 — 30/38 1
PH2 6 4 10 — 37/48 1
PH3 2 5 7 — 26/36 1
PH4 1 2 3 + 36/48 1 36/48
PH5 1 2 3 + 25/47 na 25/47
PH6 1 6 7 + 37/48 1 30/48
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a Line names correspond to those in Figure 5. b Number of plants found to be positive for or express the transgene out of the total number of T1 plants analyzed. c Number of loci determined by the presence of transgene ratios with no significant difference from that expected, as determined using a χ2 test. na, not adapted to the Mendelian segregation patterns of 3 : 1 or 15 : 1.

Inheritance of the transgenes in the T1 generation

To examine the inheritance of the integrated transgenes, we analyzed the segregation of genes among the progenies of the 20 lines used in the Southern blot analysis. We analyzed the presence or absence of the transgene sequences in T1 progenies derived from the selfed T0 plants using PCR. Of the 20 transgenic T0 lines, 18 grew to maturity, while the remaining two were sterile. The progeny of 13 of the 18 fertile lines showed a transgene segregation not significantly different from 3 : 1, indicating that the multiple transgene copies in these lines were located at a single locus (Table 2). In the T1 generations recovered from plants generated using procedure PH (lines PH4, PH5, and PH6), the segregation pattern of the GUS activity almost corresponded with those of the uidA gene sequences confirmed using PCR (Table 2). In contrast, in the T1 generations recovered from procedures C and P, only one of the five transgenic lines tested (P4) exhibited a similar segregation pattern between the uidA gene sequences and the GUS activity (Table 2).

Discussion

The previously reported efficiencies of wheat biolistic transformation are relatively low, at 0.1–5.0% (Janakiraman et al. 2002; Sahrawat et al. 2003). These transformation efficiencies were calculated using a selectable marker gene in most cases. The value of the transformation efficiency has different meanings depending on whether selectable marker genes or reporter genes are used, because the expression of a reporter gene cannot be selected. In the present study, we focused on the efficiency of transgene expression rather than the efficiency of transgene introduction.

To achieve successful transformation in wheat, the optimal preparation of explants remains of considerable interest. The LTP of explants used in the present study is likely to have two types of effect. First, LTP likely enhances tolerance for damage caused by bombardment and the osmotic stress of using a HMC during callus culture. In the present study, the efficiencies of both transgene introduction and transgene expression were lowest in plants exposed to procedure H, indicating the HMC has negative effects in wheat tissue culture. The LTP raised the efficiency of transgene introduction from 1.25 to 4.17% and eliminated the negative effect of HMC (Figure 4). We speculate that the moderate low-temperature stress adapts the cell states in the explants to the subsequent transformation and tissue culture process. This assumption is consistent with the observation reported by Pellegrineschi et al. (2002b), who showed that subjecting the donor plant to mild heat or drought stresses improves the transformation efficiency of durum wheat (Triticum durum). The second effect of the LTP is the improvement of the explant’s potential for somatic embryogenesis. Although this effect has yet to be fully elucidated, Li et al. (2003b) reported that a similar pretreatment, spike storage at 4°C, improved the somatic embryogenesis efficiency of isolated wheat scutellar tissues. They also demonstrated that the efficiency of somatic embryogenesis was dependent on sucrose concentrations in the culture medium in combination with the storage pretreatment. These findings are in agreement with our results, suggesting that a relationship exists between the pretreatment of explants and the osmotic conditions used for the tissue culture.

The integration of high-copy-number transgenes into the host genome typically leads to the unstable expression of the transgenes (Travella et al. 2005). In this report, we showed that the use of HMC (15%; 0.44 M) during the callus culture was effective in reducing the copy number of the transgenes (Table 2), which may be relevant to the observed stable expression of the transgenes. In addition, the analysis of transgene inheritance revealed that multiple copies of the transgene were typically integrated at a single locus in the wheat genome (Table 2). Thus, the HMC is considered to have suppressed the multiplication of the foreign plasmid DNA before and/or during its integration into the wheat genome. The biolistic transformation of plant cells comprises two major stages: first, foreign DNA is introduced into the plant cell nucleus, and second, the foreign DNA is integrated into the plant genome. It was suggested that, in the first stage, the recombination, ligation, and/or concatenation of the plasmid DNA molecules occurs either by a homologous recombination between common plasmid sequences or by the blunt-end ligation of linearized molecules (Gorbunova and levy 1997). These rearranged DNA molecules then integrate into the plant genome by an illegitimate recombination, typically at a few loci, and further integration events in the same locus can occur by homologous recombination (Kohli et al. 1998). We therefore hypothesize that HMC was effective in suppressing these rearrangements of the plasmid DNA during the first stage of the biolistic transformation, as well as preventing any further integration into the same locus. The suppression of rearrangements may reduce the likelihood of aberrant expression of the transgenes, leading to their stable expression.

In the present study, we determined that the stable expression of transgenes can be enhanced by two procedures during the biolistic transformation of wheat. The LTP of explants seemed to be effective in enhancing their tolerance for callus culture using HMC, which was in turn revealed to suppress the transgene copy number. When combined, these two conditions improved the efficiency of transgene expression in wheat biolistic transformation.

Acknowledgments

The authors thank Dr. Alessandro Pellegrineschi (CIMMYT) for providing the seeds of ‘MPB Bobwhite 26.’ We are also grateful to Dr. Matthew P. Perry and Dr. Wendy A. Harwood (John Innes Centre) for providing the plasmid DNA pAL156. We also thank Y. Watanuki, Y. Naito, J. Yoshihara, and K. Fujino (Institute of Crop Science, NARO) for their technical assistance. This work was supported by a grant for the ‘Development of Innovative Crops through the Molecular Analysis of Useful Genes’ from the National Agriculture and Food Research Organization, Japan, and a grant-in-aid from the Bio-oriented Technology Research Advancement Institution (Promotion of Basic Research Activities for Innovative Biosciences, No. H20/seeds-01-01), Japan.

Abbreviations

HMC

high maltose concentration

LTP

low-temperature pretreatment

References

  1. Altpeter F, Vasil V, Srivastava V, Stöger E, Vasil IK (1996) Accelerated production of transgenic wheat (Triticum aestivum L.) plants. Plant Cell Rep 16: 12–17 [DOI] [PubMed] [Google Scholar]
  2. Barcelo P, Rasco-Gaunt S, Thorpe C, Lazzeri PA (2001) Transformation and gene expression. In: Shewry PR, Lazzeri PA, Edwards KL (eds) Advances in Botanical Research 34: Incorporating Advances in Plant Pathology. Academic Press, London, pp59–126
  3. Breitler JC, Labeyrie A, Meynard D, Legavre T, Guiderdoni E (2002) Efficient microprojectile bombardment-mediated transformation of rice using gene cassettes. Theor Appl Genet 104: 709–719 [DOI] [PubMed] [Google Scholar]
  4. Brettschneider R, Becker D, Lörz H (1997) Efficient transformation of scutellar tissue of immature maize embryos. Theor Appl Genet 94: 737–748 [Google Scholar]
  5. Chen L, Zhang S, Beachy RN, Fauquet CM (1998) A protocol for consistent, large-scale production of fertile transgenic rice plants. Plant Cell Rep 18: 25–31 [Google Scholar]
  6. Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5: 213–218 [DOI] [PubMed] [Google Scholar]
  7. Fu X, Duc LT, Fontana S, Bong BB, Tinjuangjun P, Sudhakar D, Twyman RM, Christou P, Kohli A (2000) Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns. Transgenic Res 9: 11–19 [DOI] [PubMed] [Google Scholar]
  8. Gorbunova V, Levy AA (1997) Non-homologous DNA end joining in plant cells is associated with deletions and filler DNA insertions. Nucleic Acids Res 25: 4650–4657 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Harwood WA (2012) Advances and remaining challenges in the transformation of barley and wheat. J Exp Bot 63: 1791–1798 [DOI] [PubMed] [Google Scholar]
  10. Harwood WA, Ross SM, Cilento P, Snape JW (2000) The effect of DNA/gold particle preparation technique, and particle bombardment device, on the transformation of barley (Hordeum vulgare). Euphytica 111: 67–76 [Google Scholar]
  11. Hess JR, Carman JG (1998) Embryogenic competence of immature wheat embryos: Genotype, donor plant environment, and endogenous hormone levels. Crop Sci 38: 249–253 [Google Scholar]
  12. Ismagul A, Yang N, Maltseva E, Iskakova G, Mazonka I, Skiba Y, Bi H, Eliby S, Jatayev S, Shavrukov Y, et al. (2018) A biolistic method for high-throughput production of transgenic wheat plants with single gene insertions. BMC Plant Biol 18: 135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Janakiraman V, Steinau M, McCoy SB, Trick HN (2002) Recent advances in wheat transformation. In Vitro Cell Dev Biol Plant 38: 404–414 [Google Scholar]
  14. Jones HD (2005) Wheat transformation: Current technology and applications to grain development and composition. J Cereal Sci 41: 137–147 [Google Scholar]
  15. Kohli A, Leech M, Vain P, Laurie DA, Christou P (1998) Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots. Proc Natl Acad Sci USA 95: 7203–7208 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kohli A, Twyman RM, Abranches R, Wegel E, Stoger E, Christou P (2003) Transgene integration, organization and interaction in plants. Plant Mol Biol 52: 247–258 [DOI] [PubMed] [Google Scholar]
  17. Li B, Leung N, Caswell K, Chibbar RN (2003a) Recovery and characterization of transgenic plants from two spring wheat cultivars with low embryogenesis efficiencies by the bombardment of isolated scutella. In Vitro Cell Dev Biol Plant 39: 12–19 [Google Scholar]
  18. Li B, Caswell K, Leung N, Chibbar RN (2003b) Wheat (Triticum aestivum L.) somatic embryogenesis from isolated scutellum: Days post anthesis, days of spike storage, and sucrose concentration affect efficiency. In Vitro Cell Dev Biol Plant 39: 20–23 [Google Scholar]
  19. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15: 473–497 [Google Scholar]
  20. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8: 4321–4326 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pastori GM, Wilkinson MD, Steele SH, Sparks CA, Jones HD, Parry MAJ (2001) Age-dependent transformation frequency in elite wheat varieties. J Exp Bot 52: 857–863 [DOI] [PubMed] [Google Scholar]
  22. Pellegrineschi A, Brito RM, Velazquez L, Noguera LM, Pfeiffer W, McLean S, Hoisington D (2002a) The effect of pretreatment with mild heat and drought stresses on the explant and biolistic transformation frequency of three durum wheat cultivars. Plant Cell Rep 20: 955–960 [Google Scholar]
  23. Pellegrineschi A, Noguera LM, Skovmand B, Brito RM, Velazquez L, Salgado MM, Hrnandez R, Warburton M, Hoisington D (2002b) Identification of highly transformable wheat genotypes for mass production of fertile transgenic plants. Genome 45: 421–430 [DOI] [PubMed] [Google Scholar]
  24. Rasco-Gaunt S, Riley A, Barcelo P, Lazzeri PA (1999) Analysis of particle bombardment parameters to optimise DNA delivery in wheat tissues. Plant Cell Rep 19: 118–127 [DOI] [PubMed] [Google Scholar]
  25. Rasco-Gaunt S, Riley A, Cannell M, Barcelo P, Lazzeri PA (2001) Procedures allowing the transformation of a range of European elite wheat (Triticum aestivum L.) varieties via particle bombardment. J Exp Bot 52: 865–874 [DOI] [PubMed] [Google Scholar]
  26. Rogers SO, Quatrano RS (1983) Morphological staging of wheat caryopsis development. Am J Bot 70: 308–311 [Google Scholar]
  27. Sahrawat AK, Becker D, Lütticke S, Lörz H (2003) Genetic improvement of wheat via alien gene transfer, an assessment. Plant Sci 165: 1147–1168 [Google Scholar]
  28. Tassy C, Partier A, Beckert M, Feuillet C, Barret P (2014) Biolistic transformation of wheat: Increased production of plants with simple insertions and heritable transgene expression. Plant Cell Tissue Organ Cult 119: 171–181 [Google Scholar]
  29. Travella S, Ross SM, Harden J, Everett C, Snape JW, Harwood WA (2005) A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep 23: 780–789 [DOI] [PubMed] [Google Scholar]
  30. Vain P, McMullen MD, Finer JJ (1993) Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep 12: 84–88 [DOI] [PubMed] [Google Scholar]
  31. Wu H, Sparks C, Amoah B, Jones HD (2003) Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep 21: 659–668 [DOI] [PubMed] [Google Scholar]
  32. Yao Q, Cong L, Chang JL, Li KX, Yang GX, He GY (2006) Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment. J Exp Bot 57: 3737–3746 [DOI] [PubMed] [Google Scholar]

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