Journal of Experimental Botany, Vol. 52, No. 357, pp. 857-863,
April 15, 2001
© 2001 Oxford University Press
Age-dependent transformation frequency in elite wheat varieties
Biochemistry and Physiology Department, IACR-Rothamsted, Harpenden, Herts AL5 2JQ, UK
Received 17 July 2000; Accepted 8 November 2000
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Abstract |
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Wheat is a major world crop and as such is a primary target for improvement of agronomic characteristics via genetic engineering. Optimization of transformation is essential in order to overcome the relatively low transformation frequencies encountered with wheat. Transformation of elite wheat varieties is not always successful due to variability in regeneration and transformation frequencies between varieties. In this work, two elite wheat varieties with a relatively high embryogenic capacity were transformed by particle bombardment. A strong correlation between transformation frequency and the age of wheat donor plants was observed in both varieties. The mean transformation frequency rose from 0.7% to 5% when using immature embryos from old and young donor plants, respectively. This was observed in both varieties, the best bombardments achieving up to 7.3% frequency. Using explants at an optimal developmental stage from donor plants grown under environmentally-controlled conditions has improved the reproducibility of transformation efficiency of elite wheat varieties and leads to the production of apparently phenotypically normal, fertile, transgenic plants.
Key words: Bar gene, elite wheat varieties, transformation frequency, uidA gene, wheat transformation.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Wheat is one of the most important crops with annual yields exceeding 500 MT (FAO, 2000). It forms a staple part of the diet in over 60 countries, being 10–20% of the daily calorific intake and unique in its ability to leaven bread. Wheat is an important model for physiological, biochemical and genetic studies in monocotyledonous species. However, it was not until the mid-1990s that wheat transformation became routine.
Successful genetic manipulation requires the ability to deliver biologically active and functional DNA into plant cells followed by recovery of transgenic plants expressing a foreign gene. Particle bombardment is an established method for the transformation of many species, particularly cereals (Vasil et al., 1992
; Weeks et al., 1993; Becker et al., 1994; Nehra et al., 1994; Barcelo and Lazzeri, 1995; Altpeter et al., 1996; Blechl and Anderson, 1996; Stoger et al., 1998; Rasco-Gaunt et al., 1999a, b). However, optimization of the transformation protocol is essential in order to address the most frequent problems encountered in genetic manipulation of important crops, such as poor regenerability, complicated and long-term regeneration, low transformation frequency, reduced fertility, phenotypic abnormalities, altered ploidy, and loss of transgene activity in subsequent generations. Moreover, the application of this technology to elite wheat varieties is not always possible due to the variability in regeneration and frequency rates of transformation between varieties (Iser et al., 1999).
Most of the attempts to increase transformation frequency have been focused on genotype, embryogenic capacity of the explant, bombardment conditions (amount of DNA, pressure, distance, etc.), type of selection and time allowed for each stage of the procedure (Altpeter et al., 1996; Iser et al., 1999; Rasco-Gaunt et al., 1999a, 2000). Alternatively, increases in wheat transformation frequencies of individual experiments were reported by using callus derived from tissue-culture regenerated plants of two model wheat cultivars (Harvey et al., 1999). However, comparatively little attention has been paid to the developmental stage of donor plants and the environmental conditions in which they are grown. Environmental conditions are known to influence the embryogenic competence of immature wheat embryos and levels of endogenous hormones in kernels (Hess and Carman, 1998); this may significantly alter transformation frequencies.
Using explants at an optimal developmental stage from donor plants grown under environmentally-controlled conditions has improved the reproducibility of transformation efficiency of elite wheat varieties and leads to the production of apparently phenotypically normal, fertile, transgenic plants.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Plant material and growth conditions
Wheat (Triticum aestivum L.) plants were grown in environmentally-controlled chambers at 18/15 °C (day/night), with a 16/8 h photoperiod and a photosynthetic photon flux density (PPFD) of 700 µmol m-2 s-1 at a relative humidity of 80%. Plants from varieties Cadenza and Canon, previously identified for their response to tissue culture and transformation (Rasco-Gaunt, 1999
), were grown for 10–13 weeks until explants were collected for bombardment. No ears with embryos of suitable size for bombardments were available before 10 weeks (see Transformation procedure).
Gene construct
The plasmid pAHC25 (Christensen and Quail, 1996) was used for bombardment experiments. It contains the bar gene as selectable marker, which confers resistance to the herbicide L-phosphinothricin (L-PPT), and the screenable uidA gene, coding for ß-glucuronidase, both under the control of the maize ubiquitin promoter.
Transformation procedure
The transformation procedure is based on the method described previously (Barcelo and Lazzeri, 1995) and subsequently modified (Rasco-Gaunt et al., 1999a; Rasco-Gaunt and Barcelo, 1999). Scutella from immature embryos were used as explants for wheat transformation by particle bombardment. Immature caryopses were collected, surface-sterilized with 70% ethanol for 5 min, 10% hypochlorite solution for 15–20 min and rinsed twice with sterile distilled water. A total of 180–240 embryos of 0.5–1.5 mm size (Maddock et al., 1983; Rasco-Gaunt et al., 1999a) were isolated in each bombardment. Scutella were aseptically excised under a stereo microscope, placed in groups of 30 embryos per plate on a callus induction medium containing MS-medium (Murashige and Skoog, 1962) with vitamins (minus glycine), myo-inositol (100 mg l-1), the amino acids L-glutamine (375 mg l-1), L-proline (75 mg l-1) and L-asparagine (50 mg l-1), 9% sucrose, 0.5 mg l-1 2,4-D, and 10 mg l-1 AgNO3 and pre-cultured for 1–2 d at 26 °C in the darkness before bombardment (Rasco-Gaunt and Barcelo, 1999; Rasco-Gaunt et al., 2000). Two extra plates of 20 embryos each were prepared per bombardment, one for non-bombarded controls and the other for scutella bombarded with gold only.
Plasmid DNA (approximately 5 µg) was precipitated onto 0.4–1.2 µm gold particles (Bio-Rad, Richmond, CA) in the presence of 1.2 M CaCl2 and 20 mM spermidine. DNA-coated gold particles were washed once in 100% ethanol, resuspended in 75 µl of the same solution and kept on ice until bombardment. Macrocarriers were loaded with 5 µl of DNA-coated gold particles and non-DNA-coated particles were used as a control. An additional non-bombarded control was included in the experiments. Bombardments were performed using the PDS 1000/He particle gun (Bio-Rad, Munich, Germany) at an acceleration pressure of 4.75–7.6 MPa. Callus induction of bombarded and non-bombarded explants was carried out on the same medium for 4–5 weeks in darkness. The callus-induction stage was followed by regeneration of embryogenic calli in the light. The regeneration stage was performed in four rounds of 3–4 weeks each; the selection of putative transformants being applied on the last three rounds of regeneration. Regeneration (R) medium consisted of MS-medium (Murashige and Skoog, 1962), myo-inositol (200 mg l-1), thiamine HCl (10 mg l-1), pyridoxine HCl (1 mg l-1), nicotinic acid (1 mg l-1), Ca-pantothenate (1 mg l-1), L-ascorbic acid (1 mg l-1), 3% maltose, 0.1 mg l-1 2,4-D, 10 mg l-1 AgNO3, and 5 mg l-1 zeatin. Selection was carried out in R medium containing 4 mg l-1 L-PPT without added hormones and AgNO3 (Rasco-Gaunt and Barcelo, 1999; Rasco-Gaunt et al., 2000). Half of control shooted-calli were cultured in the same medium without L-PPT, the other half being used to test the efficiency of L-PPT treatment. Root development was achieved during selection and plants surviving the selective pressure (putative transgenics) were transferred to soil and grown to maturity in a glasshouse under optimal conditions. This transformation procedure allows the production of apparently phenotypically normal transgenic plants in 15–18 weeks, from the isolation of scutella throughout all tissue culture steps until plants are ready to be transferred to soil.
Regeneration capacity analysis
The embryogenic capacity of wheat scutella was estimated in non-bombarded explants as well as in those bombarded with gold only and with gold plus DNA after 4 weeks of culture on callus induction medium in the dark. The embryogenic calli were scored in a scale of 0 to 4 (P Lazzeri and P Barcelo, personal communication), each number representing a percentage range of embryogenic area: 0, 0%; 1, 1–25%; 2, 26–50%; 3, 51–75%; 4, 76–100% embryogenesis.
DNA analysis
Leaf genomic DNA from control and putative transgenic plants was extracted according to the method described earlier (Stacey and Isaac, 1994). The presence of the transgenes was checked by polymerase chain reaction (PCR) in a reaction mixture containing 10 mM TRIS-HCl pH 8.8, 50 mM KCl, 1.5 mM MgCl2, 0.1% (v/v) Triton X-100, 200 µM of each dNTP, 0.3 µM of forward and reverse primers, 50–200 ng DNA, and 0.66 units of Taq DNA polymerase (MBI, Helena BioSciences, Sunderland, UK). Primers used to detect the uidA gene were 5'-AGTGTACGTATCACCGTTTGTGTGAAC-3' and 5'-ATCGCCGCTTTGGACATACCATCCGTA-3' at an annealing temperature of 62 °C, releasing an amplified product of 1.05 kb. The bar gene was amplified with 5'-GTCTGCACCATCGTCAACC-3' and 5'-GAAGTCCAGCTGCCAGAAAC-3' primers at 57 °C-annealing temperature giving a PCR product of 443 bp.
Transgene expression analysis
Expression of the bar gene was analysed by the ammonium assay (Rasco-Gaunt et al., 1999b) which allows the quantitative and qualitative detection of phosphinothrycin acetyl transferase activity. Leaf pieces of 4x8 mm size were incubated for 5 h at 24 °C under a PPFD of 250 µmol m-2 s-1 in a medium consisting of 50 mM potassium-phosphate buffer pH 5.8, 2% sucrose, 1 mg l-1 2,4-D, 25 mg l-1 gluphosinate ammonium, and 0.1% Tween-20. The concentration of ammonium ions released to the incubation medium was estimated in a reaction mixture containing 200 µl incubation medium and 1 ml of Reagent 1 (34 g sodium salicylate GPR [BDH, UK], 25 g trisodium citrate [Fisher Scientific, UK], 25 g sodium tartrate GPR [BDH, UK] and 0.12 g sodium nitroprusside GPR [BDH, UK] per litre of deionized water) followed by the addition of 1 ml of Reagent 2 (30 g sodium hydroxide [Fisons, UK] and 0.52 g sodium dichloroisocyanurate GPR [BDH, UK] per litre of deionized water). Colour developed after 15 min incubation at 37 °C plus another 15 min at room temperature. The presence of ammonium ions resulted in an emerald green to dark-blue colour while a yellow colour reaction occurred in the absence of ammonium ions.
Transformation frequency
Frequencies of transformation were estimated as the number of transgenic plants, i.e. plants surviving L-PPT selection and PCR-positive for the bar gene, per number of bombarded embryosx100.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The embryogenic capacity of immature embryos isolated from the elite wheat varieties Cadenza and Canon was scored after 4 weeks of culture in callus induction medium in the dark (Table 1
). In this work, high regeneration frequencies were directly linked to high embryogenic capacities for both varieties. The embryogenic capacity of non-bombarded calli of both varieties was 70%. This high embryogenic capacity was maintained in bombarded tissues of Cadenza and Canon, both in controls bombarded with gold only and in treatments bombarded with gold plus DNA. There was no correlation between transformation frequency and the embryogenic capacity in either variety (r2=0.0106 for Cadenza; r2=0.0179 for Canon), suggesting that low transformation frequencies are not due to low regeneration capacity of the tissue.
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The selection of transformants was performed using regeneration medium containing 4 mg l-1 PPT after approximately 8 weeks of consecutive culture on callus induction and regeneration media. Control calli from both varieties were separated in two groups, one was treated with herbicide and the other kept as a control of regeneration capacity. Both non-bombarded calli and calli bombarded with gold only did not survive the treatment with the herbicide in the first round of selection, confirming the efficiency of PPT-selection. Plants surviving herbicide-selection, called putative transgenics, were tested for the presence of the transgenes by PCR with specific primers (Fig. 1
). The presence of the bar gene was detected in most putative transgenic plants indicating that the frequency of escapes, i.e. plants surviving the herbicide but lacking any transgene, was relatively low. In all cases, the bar gene co-integrated with the uidA gene in wheat plants from both varieties.
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The expression of the bar gene was examined in three independent transgenic lines of each variety selected by PCR analysis (Fig. 2
). All plants that were positive for the bar gene showed expression in the ammonium test, indicated by the pale green-yellow colour that results from phosphinothricin acetyl transferase activity. PPT-susceptible plants, i.e. non-bombarded controls, gave a mid to dark blue colour while leaf tissue of PPT-resistant plants, i.e. transgenic plants expressing the bar gene, showed a yellow to light green or pale blue colour. This indicates that the foreign DNA was integrated into the wheat genome and was being transcribed and translated to yield functional protein. Southern blot analysis of 20 independent transgenic lines derived from high-frequency bombardments revealed that both copy number and integration pattern of the transgene were similar to those of transgenic lines produced in low-frequency bombardments (data not shown).
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The stringency of the selection procedure was demonstrated by a low percentage of escapes, i.e. number of plants surviving herbicide-selection but lacking the transgene (data not shown). In Cadenza, 68.5% of a total of 17 bombardments resulted in escapes below 30%, and 43% of these bombardments had no escapes. Similar values were achieved in the variety Canon. From a total of 16 bombardments, 41% had no escapes. In general, the highest number of escapes corresponded to bombardments with low regeneration capacity. There was no significant correlation between percentages of escapes and transformation frequency (r2=0.512 for Cadenza; r2=0.623 for Canon) and there were no significant differences between the two varieties in terms of their ability to survive/escape the herbicide.
A high correlation between transformation frequency and the age of wheat donor plants was observed in varieties Cadenza and Canon (r2=0.946, Fig. 3). A total of 2790 immature embryos of Cadenza were bombarded in 16 separate experiments while 2950 embryos of Canon were used in 15 independent bombardments. Frequencies of transformation, expressed as the number of transgenic plants per number of bombarded embryosx100, were separated in three ranges and the age of the donor plants recorded (Table 2.). In both varieties there was a strong correlation between transformation frequency and the age of the donor plants. In Cadenza, there was an increase of 7.6 times in transformation frequency when embryos were isolated from wheat plants grown for 75 d compared to those isolated from plants grown for 82 d. Similarly, in Canon the transformation frequency was raised 7.7 times when embryos were isolated from 74-d-old plants compared with 81-d-old ones. Analysis of the availability of embryos at the optimum size for bombardment revealed that most of the embryos of the suitable size (i.e. within a range of 0.5–1.5 mm) are available from 74–84-d-old plants, where 50–70% of the ears are suitable to be collected for bombardment (data not shown). At the first stages of development, the availability of embryos results from a combination of main shoots and primary tillers. However, as time progresses the collection of suitable embryos relies exclusively on secondary tillers. Interestingly, the best bombardments achieved transformation frequencies of up to 7.1% for Cadenza and 7.3% for Canon and in both cases embryos were isolated from donor plants that had been grown for exactly 70 d. This indicates that the younger the donor plant, the better the transformation frequency will be, and that embryos collected from main shoots and primary tillers are the ones that will give high frequencies of transformation.
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Discussion |
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Wheat transformation is still a major bottleneck in the application of genetic manipulation to this important crop and improvement of the efficiency is a priority. Whether using particle bombardment, electroporation or Agrobacterium tumefaciens, wheat transformation frequencies are often around 1% and usually performed in model cultivars (Weeks et al., 1993
; Becker et al., 1994; Blechl and Anderson, 1996; Nehra et al., 1996; Altpeter et al., 1996; He et al., 1994; Cheng et al., 1997). Increased frequencies of individual transformation experiments were reported using callus derived from tissue-culture regenerated plants of two model wheat cultivars (12%; Harvey et al., 1999), however, the prolonged periods of tissue culture employed may lead to genetic instability. More significantly, increased frequencies of individual transformation experiments were also reported using immature scutella of elite wheat varieties (20.2%; Rasco-Gaunt et al., 2000). High wheat transformation frequencies (9.3%) were also obtained in model cultivar Bobwhite by using scutellum-derived calli with long-term morphogenic potential for plant regeneration, although this was limited to a small number of bombarded explants (Zhang et al., 2000). Most of the attempts to increase transformation frequency have considered genotype, embryogenic capacity of the explant, bombardment conditions, type of selection and time allowed for each stage of the procedure, resulting in improved but not reproducible efficiencies.
The use of elite varieties in the transformation of wheat is a difficult task due to, among many factors, variety-specific differences in the formation of embryogenic callus and plant regeneration (Viertel et al., 1998; Iser et al., 1999). Model wheat cultivars have a relatively high embryogenic capacity, robustness and are easily regenerable, making ‘zero’ experiments rare (Becker et al., 1994; Hess and Carman, 1998; P Lazzeri, personal communication). These properties have made them suitable for transformation for many years. Elite varieties, however, have a wide range of response to tissue culture and the efficiency of callus induction and regeneration of these varieties seems to be genotype-dependent (Fenfoldi and Purnhauser, 1992; Fennell et al., 1996; Viertel et al., 1998). This work confirms the relatively high embryogenic capacity (about 70%) observed in both varieties, Cadenza and Canon, under non-bombarded control conditions, when compared with model and elite wheat cultivars (Rasco-Gaunt et al., 2000). The high embryogenic capacity was maintained in bombarded tissues in both varieties, indicating that the stress provoked by the bombardment itself does not affect the good response of these varieties to tissue culture.
No correlation between the embryogenic capacity of immature embryos and frequency of transformation was found in Cadenza and Canon wheat varieties. The embryogenic capacity was high in both varieties (above 70%), even at low transformation frequencies. Takumi and Shimada examined six common wheat varieties for regeneration and transformation and concluded that variation in the transformation frequency was generally caused by the difference in the in vitro culture response with the genotype (Takumi and Shimada, 1997). However, Iser et al. demonstrated that there is no parallelism between the frequencies of regeneration and transformation, indicating a more fundamental genotype-dependent transformation potential (Iser et al., 1999).
In this work, it is demonstrated that the frequency of transformation of wheat genotypes is strongly dependent on the age of the donor plant. Young donor plants and, particularly, the shoots selected from these plants (i.e. ears from main shoots and primary tillers) will determine the frequency of transformation. This choice of young donor material allows the consistent production of transgenic wheat plants with a transformation frequency of 5% by using two elite varieties as donor material. Preliminary data indicate that the same correlation is applicable to three other elite wheat varieties, Avans, Riband and Imp (J West, L Rong and GM Pastori, personal communications), previously selected for their good response to tissue culture and transformation (Rasco-Gaunt, 1999). It has been reported previously that small embryos have more embryogenic potential than large embryos (Carman et al., 1988), suggesting that embryos lose embryogenic competence with age. Hess and Carman demonstrated that the dynamic hormonal regime in which embryos develop in ovulo may accelerate the loss of embryogenic capacity, and that certain environmental conditions, such as high donor plant temperatures, increase the acquisition of hormone regimes that eliminate embryogenic competence even in small embryos (Hess and Carman, 1998). It is known that the concentration of endogenous hormones during wheat embryogenesis shows a series of marked fluctuations and the relative levels of these hormones at the time of bombardment may be crucial in determining transformation potential. For example, the concentration of cytokinins in wheat ears increases to a maximum immediately after anthesis, then drops steadily to around zero at 13 d post-anthesis (DPA) (Wheeler, 1972). Gibberellins and auxins increase from low levels at anthesis to peak at 21 and 28 DPA, respectively, while the maximum concentration of ABA in developing wheat grain is at 25 d after pollination (Black, 1991). It has also been shown that high cytokinin levels during the early stages of zygotic embryogenesis and in the formation of embryogenic calli may be required for cell division (Murray, 1988; Carman, 1989). However, the ratios of endogenous auxins to cytokinins rather than their absolute levels may be more important in determining embryogenic capacity. Embryogenic competence is prolonged when embryos are exposed to low in ovulo IAA, ABA or cytokinin levels during histodifferentiation, usually 8–14 DPA (Hess and Carman, 1998). In this work, the best bombardments in both varieties were achieved by using young donor plants that had been grown for just 70 d, which corresponds to embryos at 12–14 DPA coinciding with dramatic changes in the levels of cytokinins, auxins and gibberellins in this tissue.
It is concluded that the developmental stage of donor plants is an essential factor in determining the success of wheat transformation. The concentration of endogenous hormones at the critical age of 70 d may influence the control of cell cycle, and the co-ordination of cell division and DNA replication in immature embryos, and hence, transformation efficiency. The release of senescence-related signals at a later developmental stage, as main shoots, primary tillers and the whole plant start to senesce, may contribute to block these mechanisms and decrease the ability of cells in immature embryos to integrate the foreign gene. Understanding the role that these signals play on the physiological state of embryos at the time of bombardment will permit the control of the complicated and challenging process of wheat transformation.
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Acknowledgments |
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IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council (BBSRC) of the United Kingdom. GM Pastori and MD Wilkinson acknowledge financial support from BBSRC and Food Standards Agency, respectively. The authors acknowledge Drs Paul Lazzeri and Pilar Barcelo for helpful discussions and Dr Sancha Salgueiro and Jevon West for valuable contribution in the identification and tagging of donor material.
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Notes |
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1 To whom correspondence should be addressed. Fax: +44 1582 763010. E-mail: gabriela.pastori{at}bbsrc.ac.uk
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Abbreviations |
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DPA, days post-anthesis; GUS, ß-glucuronidase; L-PPT; L-phosphinothricin; PPFD, photosynthetic photon flux density..
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References |
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