Journal of Experimental Botany, Vol. 52, No. 357, pp. 865-874,
April 15, 2001
© 2001 Oxford University Press
Procedures allowing the transformation of a range of European elite wheat (Triticum aestivum L.) varieties via particle bombardment
Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden, Hertfordshire AL5 2JQ, UK
Received 21 August 2000; Accepted 14 November 2000
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Abstract |
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Ten current European wheat varieties were transformed at efficiencies ranging from 1–17% (mean 4% across varieties) following modifications in particle bombardment and tissue culture procedures. All plants surviving phosphinothricin selection were screened for uidA and bar gene activity, and for the presence of marker gene sequences by PCR analysis. A minimum of 35% plant ‘escape’ frequency was achieved with selection on 4 mg l-1 gluphosinate ammonium after shoot initiation. Mean co-transformation frequency with various genes-of-interest was 66%. The estimated number of insertions of the uidA gene in 25 lines were; 1–2 in 32%, 3–5 in 52%, and 6–8 in 16% of lines. In T1 progenies, marker genes segregated in a Mendelian fashion in 50% of 39 lines analysed, as determined by transgene activity assays. Based on PCR analysis, it appeared that in some lines the occurrence of distorted segregation was due to poor transmission of the transgenes.
Key words: Particle bombardment, transgene integration, transgene segregation, Triticum aestivum, wheat transformation.
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Introduction |
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DNA transfer via particle bombardment is currently the standard technique applied for wheat genetic transformation, with which the modification of agronomically useful traits has now been demonstrated, for example, expression of HMW-glutenin subunit genes to modify bread-making quality (Barro et al., 1997
; Blechl et al., 1998) and the expression of stilbene synthase genes for increased fungal resistance (Leckband and Lörz, 1998). However, broad application of the technology is still limited by low and erratic stable transformation efficiencies, and by the general use of agronomically less desirable ‘model’ genotypes such as cvs Bobwhite and Florida which have been selected for their good response in tissue culture and amenability to transformation (Weeks et al., 1993; Becker et al., 1994; Altpeter et al., 1996; Blechl et al., 1998; Leckband and Lörz, 1998). The transfer of genes for improved traits to ‘model’ wheat requires subsequent transfer of the inserted transgenes into agronomically acceptable germplasm through conventional crossing and it can be difficult to separate transgenes from linkage to undesirable traits. Thus, it would be preferable if transformation technology can be applied directly to elite breeding lines.
For the application of wheat transformation in breeding programmes, it is necessary to produce numbers of transgenic lines for each gene or trait to be modified. This is because in order to be acceptable as a commercial variety, a transgenic line should satisfy a number of criteria, such as the simplicity of the transgene integration event, the level and stability of expression of the inserted gene, the stability of inheritance of the transgene, and the acceptability of the new phenotype, as well as maintaining the performance of the original line (Lazzeri et al., 1997). To find a line satisfactory in all respects, it will usually be necessary to screen a sizable population of individual transformants making transformation efficiency a fundamental consideration.
A stable transformation procedure applicable to a range of elite wheat genotypes is presented in this paper. A previous study analysed bombardment parameters by transient gene expression assays to optimize DNA delivery into wheat tissues (Rasco-Gaunt et al., 1999b). The present work aimed (a) to optimize tissue culture conditions, (b) to examine the influences of some key parameters on stable transformation, and (c) to demonstrate stable transformation of a range of elite wheat varieties using modified procedures.
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Materials and methods |
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Plant material
Wheat plants (ten current spring and winter Triticum aestivum varieties obtained from different breeders, as in Table 3
, and the model winter variety Florida) were grown in controlled environment chambers, under irradiance of c. 750 µE m-2 s-1, supplied by 400 W HQI lamps (Osram), with a 16 h light period, at 18–20 °C under lighting and 14–16 °C during darkness and 50–70% relative humidity, or in glasshouse cubicles under natural light with supplementary lighting from 400 W Son.T sodium lamps to give a minimum 16 h light period and 440 µE m-2 s-1 lighting. Imbibed seeds of winter varieties were sown into vermiculite for vernalization at 6 °C for 8 weeks, before transfer into pots containing soil mixture (Rasco-Gaunt and Barcelo, 1999).
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Isolation of scutella and culture initiation
Early–medium milk stage grains containing translucent embryos were harvested, surface-sterilized in 0.5% sodium hypochlorite for 15 min and rinsed in sterile water. Scutella were isolated and the embryonic axes removed as described earlier (Nehra et al., 1994
; Barcelo and Lazzeri, 1995). For tissue culture experiments, ten scutella were cultured per 90 mm Petri dish containing 40 ml of induction medium. For transformation experiments, 30 scutella were arranged in the centre of a 90 mm Petri dish and cultured in darkness at 25±1 °C for 1 d before bombardment.
Culture media
The basal callus induction medium, MD2 contained modified MS salts, supplemented with 3% w/v sucrose and 2 mg l-1 2,4-D (Rasco-Gaunt and Barcelo, 1999). The basal shoot induction medium, RZ contained L salts, vitamins and inositol, 3% w/v maltose, 0.1 mg l-1 2,4-D, and 5 mg l-1 zeatin D (Rasco-Gaunt and Barcelo, 1999). Regenerated plantlets were maintained in RO medium with composition as RZ, but without 2,4-D and zeatin.
Plasmid DNA and particle bombardment
The plasmid pAHC25 (Christensen and Quail, 1996) which contains the uidA gene coding for ß-glucuronidase (GUS) and the bar gene coding for resistance to phosphinothricin (PPT), both under the control of the maize ubiquitin promoter, was used in all experiments. It was delivered in co-transformations at equimolar ratio with four different plasmids: three plasmids containing S-adenosyl methionine decarboxylase (SAMDC) genes from tritordeum (Dresselhaus et al., 1996) and tomato (A Wallace, RG Fray, D Grierson, unpublished data) controlled by the same ubiquitin promoter; pUbi-Sam(–) (antisense tritordeum SAMDC), pUbi-Sam(+) (sense tritordeum SAMDC) and pUbi-Samtom (sense tomato SAMDC) and a plasmid containing an oat arginine decarboxylase (ADC) gene (Bell and Malmberg, 1990) controlled by the endosperm specific HMW glutenin 1DX5 subunit promoter; pHMW-ADC (sense oat ADC). Plasmid DNA was prepared using Qiagen Mega Kit and was precipitated onto submicron gold particles (0.6 Micron Gold, Bio-Rad) as described previously (Barcelo and Lazzeri, 1995). Particle bombardments were carried out using a PDS 1000/He gun with a target distance of 5.5 cm from the stopping plate at helium pressures between 650 and 1100 psi.
Tissue culture and selection of transformants
For tissue culture experiments, scutella were cultured on induction medium in darkness for 3 weeks at 25±1 °C for the induction of somatic embryos. For transformation, bombarded explants (30) were distributed over the surface of the medium in the original dish and two other dishes (10 explants/dish) and cultured at 25±1 °C in darkness for 3 weeks. For shoot induction in tissue culture and transformation experiments, cultures bearing somatic embryos were transferred to RZ medium and cultured under 12 h light (250 µE s-1 m-2, from cool white fluorescent tubes) at 25±1 °C for one or two 3-week rounds. In transformation experiments, successive 3-week rounds of selection in plant maintenance medium (R0) were applied until all control plantlets were killed and surviving plantlets have developed good root systems. All plants regenerating from the same scutellum/callus were noted and considered clonal plants until shown otherwise by Southern analysis. Putative transgenic plantlets were then potted in soil after 6–9 weeks in plant maintenance/selection medium. Selection on 2 or 4 mg l-1 gluphosinate ammonium (Greyhound, UK) was compared with selection on 3 mg l-1 bialaphos (Meiji Seika Kaisha Ltd, Japan).
Analyses of marker gene activity
GUS expression was assayed in young leaf tissues of regenerated T0 plants and T1 progeny plants as described earlier (Barcelo and Lazzeri, 1995), with the modification that chlorophyll was extracted after staining by incubation in 70% ethanol for 1 h followed by 100% ethanol overnight. bar gene expression was assessed by an ammonium-evolution assay (Rasco-Gaunt et al., 1999a).
Molecular analyses
DNA was isolated from leaf tissue using a CTAB procedure (Stacey and Isaac, 1994). PCR analysis was performed on 1 µl DNA [50–100 ng µl-1] in a 30 µl reaction volume containing 50 mM KCl, 10 mM TRIS-HCl (pH 8.8), 1.5 mM MgCl2, 0.1% Triton X-100, 200 µM of dNTPs, 0.3 µM of each primer, and 0.66 U of Dynazyme DNA polymerase (Flowgen, UK). Amplifications of the uidA gene (5'-AGTGTACGTATCACCGTTTGTGTGAAC-3', 5'-ATCGCCGCTTTGGACATACCATCCGTA-3', annealing temperature 62 °C), bar gene (5'-GTCTGCACCATCGTCAACC-3', 5'-GAAGTCCAGCTGCCAGAAAC-3', annealing temperature 57 °C), pUbi-Sam(-) samdc gene (5'-GACTCGGATAGCACATACGA-3', 5'-CCATCTCATAAATAACGTCATGC-3', annealing temperature 55 °C), pUbi-Sam(+) samdc gene (5'-TGCTTCGAGAATGTGGAGAGC-3', 5'-CCATCTCATAAATAACGTCATGC-3', annealing temperature 55 °C), pUbi-Samtom samdc gene (5'-CAGCAGGTCCGAGAATTTC-3', 5'-GTCGATGCTCACCCTGTTGT-3', annealing temperature 57 °C), pHMW-ADC adc gene (5'-TACTGGGGCATCCAGCATCT-3', 5'-CTTCTTACCTTGCACAGGGC-3', annealing temperature 57 °C) were made and the products analysed by electrophoresis on 0.8% w/v agarose gels. PCR product lengths were as follows; bar, 420 bp; uidA, 1020 bp; pUbi-Sam(+) samdc, 490 bp; pUbi-Sam(-) samdc, 430 bp; pUbi-Samtom samdc, 310 bp; adc, 1300 bp. Thermocycling conditions were as follows; for 30 cycles, denaturation at 94 °C for 30 s, annealing for 30 s, extension at 72 °C for 2 min.
Southern blot analysis of the uidA gene in 25 transgenic lines was subsequently performed on genomic DNA digested with the enzymes SacI and HindIII. Digested DNA was separated by electrophoresis in 0.7% w/v agarose gels and transferred to positively-charged nylon membrane (Boehringer Mannheim, Germany). Filters were hybridized with PCR-generated digoxygenin-labelled probes produced using the uidA primers for amplifying fragments internal to the gene coding region. Hybridization and chemiluminescent detection of probes was carried out according to the DIG System User's Guide for Filter Hybridization (Boehringer Mannheim, Germany).
Statistical analyses
Least Significant Difference (LSD) pairwise comparison of means was used to determine the significant differences between treatments following analysis of variance. The Chi square test (
2) was used to confirm the probability that ratios conformed to the expected Mendelian segregation patterns of 3:1 or 15:1.
Experimental design and parameters tested
In tissue culture experiments, 30–50 scutella were cultured per treatment and each experiment used two wheat varieties, contrasting in their response in culture. The frequency of somatic embryogenesis was scored at 3 weeks, and the frequency of shoot regeneration assessed after 3 and 6 weeks on regeneration medium. The proportion of embryogenic to non-embryogenic tissues per callus (embryogenesis score) and the frequency of root production were also scored, at 3 and 6 weeks, respectively.
The following factors were investigated for their influence on embryogenesis and regeneration: scutellum size (classes <0.5, >0.5–0.75, >0.75–1, >1–1.5, >1.5–2 mm); induction medium sucrose content (3, 6, 9% w/v); induction medium 2,4-D content (0.5, 1, 2, 4, 5 mg l-1); silver nitrate in induction and regeneration medium (10 mg l-1); Petri dish seal (clingfilm, Parafilm (American National Can, USA), Nescofilm (Bando Chemical Ind. Ltd., Japan), Albupore tape (Fisch Laboratoires, France), Micropore surgical tape (Boots Company, England)); induction medium MS macrosalt concentration (1/2x, 1x, 2x); regeneration medium zeatin content (5, 10 mg l-1); regeneration medium Thidiazuron content (0.1, 1 mg l-1); induction and regeneration medium cefotaxime content (60, 100 mg l-1); induction medium acetyl salicylic acid content (5, 10, 30 mg l-1); light (70 µE s-1 m-2, from cool white fluorescent tubes) or darkness during somatic embryo induction.
In transformation experiments, the following factors were investigated for their influence on transformation efficiency: bombardment pressure (650, 900, 1100 psi); amount of gold per bombardment (60, 120 µg), induction medium sucrose content (3, 6, 9% w/v); induction medium 2,4-D content (0.5, 2 mg l-1). 1–8 varieties were used for each factor tested. Transformation efficiencies were calculated on the basis of independent transgenic lines confirmed by PCR analysis. Results are presented as means for several varieties.
In a first series of experiments (Phase I procedures), the different factors were examined independently as variants of the standard procedure (Barcelo and Lazzeri, 1995), and subsequently the optimal conditions derived from these experiments were combined to give Phase II procedure which was applied to the range of genotypes. The conditions comprising the standard procedure were: 294 ng DNA load per bombardment, 120 µg macrocarrier gold (Heraeus 0.4–1.2 µm) load per bombardment, 1100 acceleration pressure, 27 in. Hg chamber vacuum pressure, and 3% sucrose and 2 mg l-1 2,4-D in the callus induction medium for the culture of target tissues.
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Results |
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Tissue culture experiments
Of 11 tissue culture variables tested, the five which had the most important effects on somatic embryogenesis and shoot regeneration are presented in Table 1
. Scutellum size strongly influenced culture response in both varieties tested, with the highest embryogenesis and shoot regeneration frequencies being observed from scutella in the 0.75–1.0 mm size class (Fig. 1a). Scutella smaller than 0.5 mm or larger than 1.5 mm were clearly less productive than those from medium size classes.
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Increasing the sucrose content of induction medium from 3% to 6% or 9% increased embryogenesis in cv. Florida (from 78% to 98%), but had less effect in cv. Riband, while in both varieties subsequent shoot regeneration was markedly improved (72% to 90%, cv. Florida; 48% to 62% cv. Riband) in cultures induced at higher sucrose concentrations (Fig. 1b
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Varying the concentration of 2,4-D in the induction medium affected embryogenesis differently in the two varieties tested. In cv. Avans a similar response was seen over the range 0.5–2 mg l-1, followed by decreased embryogenesis at 4 and 5 mg l-1, while cv. Soissons appeared more sensitive to 2,4-D and showed a stepwise decline in embryogenesis from the optimal concentration of 0.5 mg l-1. Scutella cultured on 0.5 or 1 mg l-1 2,4-D had the highest proportion of embryogenic to non-embryogenic tissue and also produced most roots (data not shown). In both varieties shoot regeneration was most efficient from cultures induced on low 2,4-D concentrations. This effect was striking in cv. Soissons, where regeneration was more than doubled by the step from 2 to 1 mg l-1 2,4-D. In cv. Avans, the highest shoot density per scutellum was observed at 0.5 mg l-1 2,4-D (Fig. 1c).
Incorporation of silver nitrate in callus induction and regeneration media increased embryogenesis in both cultivars tested. Silver nitrate also increased embryogenesis score, i.e. the proportion of embryogenic to non-embryogenic tissue per callus (data not shown). In cv. Brigadier the compound had a very marked effect on regeneration, improving the frequency from 3% to 23% (Fig. 1d). Cadenza, however, did not show a clear improvement as regeneration was generally poor.
Somatic embryogenesis from scutella was not influenced significantly by the type of sealing film used to close Petri dishes. Shoot regeneration was affected, however, with Nescofilm giving the highest frequencies in both genotypes tested (Fig. 1e). Dishes sealed with Nescofilm also showed maximal root production in both varieties (data not shown).
Among the other tissue culture variables examined, macrosalt concentration, cefotaxime, and acetyl salicylic acid did not significantly influence culture response (data not shown). Similarly, replacing the 5 mg l-1 zeatin used in standard regeneration medium with thidiazuron or 10 mg l-1 zeatin did not affect regeneration frequency (data not shown). Thidiazuron, however, influenced density of shoot formation in variety Brigadier (Fig. 1f). Incubating scutellum cultures in light during the induction of embryogenesis appeared to accelerate embryo development, but the frequency of embryogenesis and shoot regeneration were not significantly affected (data not shown).
Transformation experiments
Four factors were tested for their influence on the stable transformation of elite wheat genotypes (Table 2). Comparing three bombardment pressures, transgenic plants were only recovered at 650 and 900 psi. The effect of the amount of gold used for bombardment (60 or 120 µg) was less clear-cut, with transformation efficiencies of 0.5% and 0.6%, respectively.
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Sucrose and 2,4-D concentration in the callus induction medium both influenced stable transformation efficiency. Comparing media containing 3, 6 and 9% sucrose, transformants were recovered only from the 9% treatment at a mean efficiency of 1%. From the comparison of two levels of 2,4-D (0.5 and 2.0 mg l-1), 0.5 mg l-1 of the auxin resulted in better recovery of transgenic plants.
Transformation of elite wheat varieties
Initial experiments on elite varieties, using the original transformation procedure as developed for model wheat genotypes (see Materials and methods), gave transformation efficiencies between 0.2% and 0.7% in six cultivars, while no transformant was produced in two cultivars, giving a cultivar mean efficiency of 0.4% (Table 3, column 5). Modification of culture and bombardment parameters based on the experiments described above lead to an increase in transformation efficiency in all varieties tested (mean 10-fold increase, see Table 3, column 6) and allowed the recovery of plants from two varieties not previously transformed. With this procedure, the mean transformation efficiency over ten varieties was c. 4% and the best individual experiments in the cultivars Avans and Canon had efficiencies of 9.5% and 20.2%, respectively. In total, 21/32 experiments (66%) produced transgenic plants. The size of experiments based on the number of scutella isolated ranged from 100–400.
Analyses of primary transformants
All plants surviving PPT selection were screened; (1) for marker gene activity and (2) for the presence of marker gene sequences by PCR analysis. Lines which did not show PCR products for both marker genes (1020 band for uidA, 420 bp band for bar) were considered ‘escapes’ and discarded.
Comparing the efficiency of PPT selection systems across several experiments, the highest frequency of escapes (51/61=83.6%) was seen with the use of 2 mg l-1 gluphosinate ammonium, while at 4 mg l-1 the frequency was reduced to 35.3% (30/85). The use of 3 mg l-1 bialaphos gave an escape frequency intermediate between the high and low gluphosinate ammonium values (30/52=57.7%).
All lines were produced using the plasmid pAHC25 containing both uidA and bar genes. As expected, the number of lines with detectable GUS and PAT activity was less than the number of lines showing PCR products for the genes (Table 4). This was most marked for the GUS histochemical assay from which only 69% of plants showing a uidA gene PCR product showed GUS activity. However, it was noted that the PCR analyses for the uidA gene do not amplify the entire gene sequence.
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PCR-positive lines for either or both marker genes were further analysed for the presence of the co-bombarded gene-of-interest (samdc or adc genes). Mean co-transformation efficiency was 67% across ten varieties (Table 4
). On a per gene construct basis, co-transformation frequencies were 64% (23/36) for Ubi-Sam(+), 70% (14/20) for Ubi-Sam(-), 64% (7/11) for Ubi-Samtom, and 87% (18/21) for HMW-ADC.
Southern blot analyses were performed on 25 PCR-positive lines. Genomic DNA isolated from each line was digested with SacI, an enzyme which cuts plasmid pAHC25 once, and with HindIII to release the 4.2 kb uidA expression cassette (gene coding region and Ubi promoter). Southern blots were probed with a 1020 bp labelled uidA coding region fragment. Figure 2 shows Southern blot analysis of six primary transformants of two wheat varieties Canon (Fig. 2a) and Riband (Fig. 2b). The detection of a 4.2 kb band in DNA digested with HindIII (releasing digest=R) is shown in all 12 lines with the exception of Canon lines 2 and 5 (Fig. 2b), suggesting the presence of at least one intact uidA expression cassette. The presence of bands smaller and larger than 4.2 kb are evidence for truncations and rearrangements in the gene fragment. The detection of hybridizing fragments in DNA digested with SacI (linearizing digest=L) estimates the number of insertions of the uidA coding region. Of 25 lines analysed, the estimated number of insertions of the uidA gene were; 1–2 in 32% (8/25), 3–5 in 52% (13/25), and 6–8 in 16% (4/25) of lines (Table 4). Six of the 25 lines (24%) showed single uidA insertions. These single uidA insertion plants may, however, contain multiple insertions from other regions of the plasmid which are not expected to be revealed by the analyses conducted in this study.
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Analyses of T1 progeny lines
Ten to 35 T1 progeny plants of each of 45 primary transformants were grown to maturity. T1 progenies of 39 (of 45) primary transformants were analysed for uidA and bar gene activity. Around 50% of the lines segregated in a Mendelian fashion based on marker gene activity (Table 4). In lines in which segregation of both marker genes was examined, both genes typically exhibited the same segregation pattern. Levels of expression of the uidA gene frequently appeared weak in comparison to levels observed in primary transformants.
To determine whether aberrant segregation in 50% of the population was due to poor expression or transmission of the genes, five lines showing Mendelian and five showing non-Mendelian segregation were selected at random and analysed for the presence of the marker gene sequences by PCR. Results showed that in all ten lines, the absence of uidA and bar gene activity corresponded with the absence of the gene sequences.
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Discussion |
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The approach taken to establish a procedure for the stable transformation of elite wheats was, first, to optimize gene delivery conditions to minimize tissue injury and maximize tissue response (Rasco-Gaunt et al., 1999
b), and second (present work), to improve culture conditions for embryogenesis and shoot regeneration. By modifying several basic tissue culture parameters, regeneration from elite wheat varieties was improved. This was necessary because, under the standard culture conditions, somatic embryogenesis and, particularly, shoot regeneration from scutellar tissues of the elite wheats were poor compared to the model variety ‘Florida’. Eleven tissue culture variables were examined, among which scutellum size, callus induction medium sucrose and 2,4-D content, addition of silver nitrate to callus induction and regeneration medium, and Petri dish seal were found to influence significantly culture response, based on experiments comparing the responses of two contrasting genotypes (weakly versus strongly regenerating varieties).
The second stage was to conduct small-scale experiments to test the influence of a few parameters on the stable transformation of selected varieties. From these experiments, lower gold particle acceleration pressures, increased sucrose concentration and lower 2,4-D levels in induction medium were found to improve the recovery of transformants.
Some of the parameters tested have previously been studied individually in cereal tissue culture or transformation experiments. Osmotic treatment of target tissues for stable transformation results in plasmolysis of cells, limiting damage by preventing extrusion of the protoplasm from bombarded cells (Vain et al., 1993). Short-term high osmotic treatments, typically a few hours before or after bombardment, have been used in maize (Vain et al., 1993) and wheat (Zhou et al., 1995; Altpeter et al., 1996; Ortiz et al., 1996). Typically, non-metabolizable osmotica such as mannitol, sorbitol and PEG have been used at concentrations ranging from 0.25–1 M. In contrast, in the present study, scutella were cultured on high sucrose levels (9%; 0.27 m) for the whole callus induction period of 3 weeks. This treatment improved somatic embryogenesis, shoot induction, and post-bombardment shoot regeneration and, consequently, stable transformation efficiency.
Silver nitrate has been shown to promote callus formation and shoot regeneration in maize (Stacey and Isaac, 1994) and, in a parallel study to the present work, in wheat (Leckband and Lörz, 1998). Ag+ is a potent inhibitor of ethylene action in plants (Beyer, 1976), and ethylene inhibits somatic embryogenesis and shoot primordium formation in callus cultures by interfering with incorporation of ethylene at its receptor site. It is well known that the auxin 2,4-D, commonly used for callus induction, strongly promotes endogenous ethylene production (Yang and Hoffman, 1984). Thus, in addition to improving tissue culture response, Ag+ may act by inhibiting stress-induced ethylene production in targeted tissues.
It has previously been suggested that, in wheat bombardment, high gold particle loads suppress culture development (Becker et al., 1994; Altpeter et al., 1996), but a comparative stable transformation experiment had not been performed. Although it has been shown previously that gold load affects shoot regeneration in wheat (Rasco-Gaunt et al., 1999b), in the present work, 60 or 120 µg gold loads gave similar stable transformation efficiencies.
Results from the experiments on tissue culture and bombardment parameters and previous transient expression studies were used as the basis for modifying the standard transformation procedure (Barcelo and Lazzeri, 1995) one developed for the transformation of amenable cereal species such as tritordeum and, ‘model’ wheats.
The final stage of the study involved conducting larger-scale transformations in which a total of 88 independent lines were regenerated from ten elite wheat varieties with mean transformation efficiencies ranging from 1–17.3% (mean 3.9±1.6% across varieties) using the modified procedure. The best efficiency attained in a single experiment was 20.2%, indicating the high potential of the system. All plants but two were fertile and morphologically normal. There have been very few previous reports of transformation of elite wheat germplasm, and mean efficiency across genotypes obtained in the present study is higher than those previously reported for ‘model’ genotypes Bobwhite and Florida (0.2% (Weeks et al., 1993); 1.3% (Becker et al., 1994); 0.15% (Zhou et al., 1995); 1.5% (Altpeter et al., 1996), and other wheat genotypes (cv. Fielder 1.2% (Nehra et al., 1994); lines L88-6 and L88-31 0.9% (Barro et al., 1997); cvs Akadaruma, Norin 12, Haruhikari, and Chinese Spring 0.8% (Takumi and Shimada, 1997), and Australian varieties (0.25–1.2%, Wirtzens et al., 1998)). Since the present experiments were completed, an additional paper by Iser et al. was published which shows transformation across four German wheat varieties at efficiencies ranging from 0.2–1% (Iser et al., 1999). The present results demonstrate the potential for efficient transformation of elite wheat germplasm, but experiment-to-experiment variation remains a difficulty for routine production of transgenics. A component of this variation derives from inherent variability in the target explants. A recent study has shown that the use of explants from younger donor plants improves transformation efficiency and reproducibility (Pastori et al., 2001).
In the present study, co-transformation efficiency of genes-of-interest with marker genes ranged from 64–87%. This compares with efficiencies of 71% and 90% of HMW subunit genes reported previously (Barro et al., 1997; Blechl et al., 1998). In the current work, genes-of-interest and marker plasmids were delivered at equimolar ratios, it is possible that co-transformation efficiencies could be further improved by supplying genes-of-interest in excess.
In the comparison of selection under different PPT regimes, the frequency of ‘escapes’ varied considerably from experiment-to-experiment, but 4 mg l-1 gluphosinate ammonium clearly gave the lowest value (35.3%). There appeared to be little difference between bialaphos and gluphosinate ammonium for selection, but it would be useful to examine both agents at higher concentrations. It is difficult to compare the present selection efficiencies with those previously reported in wheat, as in the present procedure, selection was applied ‘late’, i.e. after one or two rounds in regeneration medium in contrast to ‘early’, for example, during callus induction and early stages of regeneration. Selection was applied during later stages of regeneration in order to maximize the regeneration potential of calluses.
The majority (84%) of the 25 transgenic lines analysed by Southern blotting had uidA gene insertions between 1–5; from which 24% (6/25) contained a single insertion, 40% (10/25) contained between 1–3 insertions, and 36% (9/25) contained 4–5 insertions. Previous results in transgenic wheat obtained via particle bombardment are comparable with these data. Blechl et al. reported 25% single, 25% between one and three, 38% four or five, and 12% greater than five insertions of HMW subunit genes (Blechl et al., 1998), while, Barro et al. reported 14% of lines containing single copy, 14% between one and three, 43% with four or five and 43% with greater than five insertions of HMW-subunit genes (Barro et al., 1997). The frequency of lines with low insertion number from bombardment experiments is less than in transgenic lines derived by Agrobacterium-mediated transformation (Cheng et al., 1997) in which 35% (9/26) of lines contained a single insertion, 85% contained between 1–3 insertions and 15% had four or five insertions. None of the Agrobacterium transformation-derived lines contained more than five insertions. Moreover, a smaller proportion of these transgenic lines (50% of 39 T0 lines) as compared with 68% (34/50) (Cheng et al., 1997) segregated in a Mendelian fashion.
PCR analysis in the present work showed that in ten randomly-chosen lines (5 Mendelian and 5 non-Mendelian segregating lines), uidA and bar gene activity corresponded to the presence of the gene sequences and that lack of expression in a few lines correlated with the absence of the transgene. Thus, the deficit of ‘positive’ plants was due to poor transmission rather than poor expression or silencing of transgenes.
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Acknowledgments |
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IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom. S Rasco-Gaunt, A Riley and M Cannell were supported by BBSRC (CWIS Grant CW107196) and MAFF (Grant CE0124). The authors acknowledge Fiona Gilzean for care of plants and Sue Steele for assistance in tissue culture and transformation work. The experiments described in this article were undertaken in the UK and comply with current UK law.
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Notes |
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1 To whom correspondence should be addressed. Fax: +44 1582 768791. E-mail: Sonriza.Rasco\|[hyphen]\|Gaunt{at}gbr.dupont.com
2 Present address: DuPont Wheat Transformation Laboratory, c/o Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, UK.
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Abbreviations |
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ADC, arginine decarboxylase; 2,4-D, 2,4-dichlorophenoxyacetic acid; GUS, ß-glucuronidase; HMW, high molecular weight glutenin subunit gene; PAT, phosphinothricin acetyl transferase; PPT, phosphinothricin; SAMDC, S-adenosyl methionine decarboxylase.
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References |
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