Journal of Cereal Science 41 (2005) 137147 www.elsevier.

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Review

Wheat transformation: current technology and applications to grain development and composition
Huw D. Jones*
CPI Division, Rothamsted Research, West Common, Harpenden, Hertfordshire AL5 2JQ, UK Received 24 June 2004; revised 2 August 2004; accepted 2 August 2004

Abstract Transgenesis is a powerful research tool that can be adapted to investigate many aspects of gene function. It has been used widely in model plants such as Arabidopsis, tobacco and rice but until recently, bottlenecks in DNA-delivery and tissue culture meant that it could not be used routinely for wheat research. However, many aspects of grain development and composition are unique to wheat and cannot be easily investigated in model species. Over the last decade, progress in biolistic- and Agrobacterium-mediated DNA delivery, reduction in genotypedependency in wheat tissue culture and in the development of a range of supplementary technologies has enabled its application in this traditionally recalcitrant crop. The use of genetic modication has already made a signicant impact on our understanding of interactions between high molecular weight glutenin subunits and their individual contribution to dough strength. As candidate genes become available the application of genetic transformation is set to play a major part in the elucidation of their function in determining other important grain traits such as starch and lipid composition, dietary bre composition and grain texture. q 2004 Elsevier Ltd. All rights reserved.
Keywords: Wheat; Transformation; Genetic modication; Endosperm; Agrobacterium

1. Introduction Wheat is grown on approximately 17% of the worlds cultivatable land (over 200 million hectares) and is one of the most important sources of calories and protein in the human diet. It also supplies essential vitamins and minerals such as vitamins B and E, magnesium and phosphorous, as well as bre. It is the only cereal which produces gluten allowing the production of leavened bread and is the major ingredient in other goods such as biscuits and cakes, pasta, noodles and some breakfast cereal products. In addition, wheat-derived ingredients are added to a wide range of processed foods to confer specic functional properties. Low-grade wheat and industrial wheat by-products are used for animal feed. Despite its global importance, wheat was the last major cereal to be genetically transformed. It is only a decade since the recalcitrance of this crop to in vitro culture was
Abbreviations: BAP, benzyladenine purine; HMWG, high molecular weight glutenin; AMI, phosphomannoisomerase; PPT, phosphinothricin. * Tel.: C44 1582 763 133; fax: C44 1582 763 010. E-mail address: huw.jones@bbsrc.ac.uk 0733-5210/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2004.08.009

overcome and the rst reports of fertile adult transgenic wheat plants were published (Becker et al., 1994; Nehra et al., 1994; Vasil et al., 1992, 1993; Weeks et al., 1993). Since then, other key advances have been made, including the development of Agrobacterium-mediated transformation, the reduction of genotype dependency and improvements in transformation efciencies. In addition, supplementary technologies rst developed in model species can now be applied to wheat, such as epitope or uorescent tagging of proteins, the use of tissue-specic or inducible promoters to target expression and precise RNAi-mediated down regulation. Despite the fact that bread wheat has a complex, hexaploid genome of 1.6!1010 base pairs which is ve times the size of the human genome and 35 times that of rice, transformation can be successfully used to manipulate and study individual genes involved in grain development and composition. This review summarises the current state of wheat transformation and associated technologies and highlights some of the current research utilising transgenic approaches to investigate specic grain quality traits in wheat.

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2. Transformation and tissue culture The production of fertile transgenic wheat plants depends on two processes. Firstly, donor DNA sequences must be stably integrated into the genome of a host cell (transformation) and secondly, a fertile plant must be grown via in vitro culture from this cell. A range of wheat explant types and Agrobacterium or direct gene transfer methods have been used but only a few specic combinations have been successful and these are discussed below. The recalcitrance of wheat to regeneration via tissue culture was a major limiting factor in the initial development of routine wheat transformation methods. For instance, early successful transformation of maize and rice protoplasts by electroporation or calcium/polyethylene glycol treatment (Rhodes et al., 1988a,b; Shimamoto et al., 1989) were not reproducible in wheat. The difculty of maintaining embryogenic suspension cultures to produce protoplasts and the technical challenges of regenerating wheat plants from transformed protoplasts mean that today, this approach is used only for specialist, high throughput screening of transient expression. 2.1. Explant choice Adult, fertile transgenic wheat plants can be regenerated from only a few target cell-types, although the choice is wider for transient expression studies or if transformed but non-regenerable cell lines are the nal product. Only two wheat explant tissues are currently used routinely for making transgenic plants; the immature inorescence and the scutellum of immature zygotic embryos. Other tissues that have been be used for in vitro regeneration of plants but are not yet capable of reliably producing fertile adult transgenic wheat plants include shoot meristems (Ahmad et al., 2002), leaf bases (Wang and Wei, 2004), microspores (Folling and Olesen, 2001; Ingram et al., 1999; Kunz et al., 2000; Liu et al., 2002) and mature seeds (Ozgen et al., 1998; Zale et al., 2004). The most commonly used explant for wheat transformation is the immature scutellum, a specialised tissue that forms part of the seed embryo. It is amenable to both biolistics and Agrobacterium-mediated DNA delivery methods and can be readily induced to form embryogenic callus. The optimal stage of caryopsis development for embryo isolation can differ between genotypes but is around 1116 days post anthesis (Pastori et al., 2001). A slight improvement in transformation was observed when scutella were isolated from donor plants that had themselves been regenerated via embryogenesis and tissue culture (Harvey et al., 1999). This improvement in overall transformation rates was attributed to an improvement in the consistency of donor plants resulting in fewer failed experiments. More recently, immature inorescences have been reported as an alternative source of explant for stable transformation of durum wheat (He and Lazzeri, 1998;

Lamacchia et al., 2001), tritordeum (a fertile amphidiploid between bread or durum wheat and the wild barley Hordeum chilense) (Barcelo et al., 1994; He et al., 2001) and bread wheat (Rasco-Gaunt and Barcelo, 1999; Sparks et al., 2001). This explant has also shown good expression of T-DNAdelivered genes after Agrobacterium co-cultivation (Amoah et al., 2001). Advantages of the immature inorescence system are that they are easier to isolate and are harvested from much younger plants allowing more efcient use of space in donor-plant growth facilities. However, their tissue culture response is more genotype-specic than that of immature scutella, with some varieties being particularly unresponsive (Rasco-Gaunt and Barcelo, 1999). 2.2. DNA delivery methods 2.2.1. Direct gene transfer Many DNA-transfer methods have been tried with varying degrees of success including electroporation, micro-injection, silicon carbide bres, polyethylene glycol and laser-mediated uptake, but two methods now predominate: transformation via particle bombardment and via Agrobacterium. Particle or microprojectile bombardment (also called biolistics) involves the adsorption of plasmid or linear forms of naked DNA onto the surface of submicron particles of gold or tungsten which are driven at high velocity into recipient plant cells using an acceleration device (Sanford, 1988; Sanford et al., 1993). The heliumdriven particle delivery system rst developed by DuPont and subsequently marketed by BioRad as the PDS1000/He has been widely used for this purpose, but other devices such as the particle inow gun and the ACCELLe electrical discharge technology have also been used successfully. Biolistics has also been used to deliver DNA into the chloroplast and mitochondrion genomes (for review see Sanford et al., 1993) and effective DNA-transfer has also been demonstrated using Escherichia coli or Agrobacterium cells as micro-projectiles (Rasmussen et al., 1994). Particle bombardment effectively distributes DNA over a wide area of the target tissue and is relatively genotype independent. However, several parameters must be optimised for particular explants including the microprojectile type, size and quantity; DNA quantity and method of precipitation; and the acceleration device parameters such as propellant force, helium pressure and target distance. All of these parameters can inuence the efciency of DNA delivery and the extent of damage to the explant tissues (Altpeter et al., 1996; Harwood et al., 2000; Ingram et al., 1999; Perl et al., 1992; Rasco-Gaunt et al., 1999). Although biolistic DNA delivery forms the basis of many robust and well used wheat transformation protocols (Sparks and Jones, 2004), it often leads to complex transgene integration patterns which can cause problems in subsequent analysis (discussed below). This has been a driving force in the development of Agrobacterium-mediated wheat transformation methods.

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2.2.2. Agrobacterium tumefaciens mediated delivery The soil pathogen A. tumefaciens is the causative agent of crown gall disease and is well adapted for transferring a small segment of its DNA (T-DNA) to its host plant cell. A major advance in the application of Agrobacterium to plant genetic engineering was made in the early 1980s when the large, tumour-inducing (Ti) plasmid of Agrobacterium was modied into the more easily-manipulated disarmed binary vector system in common use today (Bevan, 1984; Hoekema et al., 1983). These vector systems comprise two plasmids, one with a convenient multiple cloning site anked by T-border sequences, a selectable marker gene and an origin of replication for easy maintenance in E. coli and another, disarmed Ti plasmid, lacking the tumourinducing genes but retaining the vir loci whose products interact with the T-strand and facilitate DNA transfer to the plant cell. Cereals are not natural hosts for Agrobacterium species and research was needed to match host strains, plasmids, selection systems, wheat genotypes and media compositions (reviewed by Cheng et al., 2004). To date there are relatively few reports describing successful regeneration of adult wheat plants containing T-DNA insertions and these are summarised in Table 1. Transformation efciencies (number of independently transformed plants per 100 explants infected) reported for Agrobacterium transformation are generally higher for the model, highly regenerable wheat genotype Bobwhite than for commercial wheat germplasm, but currently lower than the best published values for biolistics. Examples of reported average transformation efciencies include 4.4% for 3354 Bobwhite lines (Hu et al., 2003), 1.8% for 44 Florida and Cadenza lines (Wu et al., 2003) and 1.9% for 17 Verry-5 lines (Khanna and Daggard, 2003). With the exception of Hu et al. (2003), these transformation rates were calculated from a relatively small number of experiments and, as with the early reports of biolistic

transformation, they are expected to increase as protocols are further developed and optimised. Agrobacteriummediated DNA-delivery is already the method of choice in many major commercial laboratories and, except for some specialised applications, is set to displace biolistics for routine wheat transformation over the next ve years. 2.3. Effect of DNA-delivery methods on transgene integration Regardless of the method used to deliver DNA into the nucleus, the process of transgene integration into the plant genome appears to be similar. Molecular analysis of integration sites suggests that transgenes insert via doublestranded illegitimate recombination, utilising the plants own DNA repair machinery, at a single (or sometimes more that one) locus. The locus may contain one or multiple, concatameric copies of the transgene, which may have undergone rearrangements and/or may have generated short lengths of ller DNA homologous to anking plant genomic DNA (Kohli et al., 1998; Pawlowski and Somers, 1998; Svitashev et al., 2000, 2002). Plants with fewer transgene copies and no rearrangements are desirable as these are easier to characterise at the molecular level, more likely to demonstrate predictable Mendelian inheritance ratios and are less likely to be targeted by silencing mechanisms, but the frequency of single copy, un-rearranged insertions in particle bombardment-generated wheat plants is rare (Kohli et al., 2003; Pawlowski and Somers, 1996, 1998). Agrobacterium is perceived to have advantages over other forms of DNA delivery methods, including biolistics, because it can introduce larger segments of DNA with minimal rearrangement and with fewer copies of inserted transgenes at higher efciencies and at lower cost (Gheysen et al., 1998; Hansen and Wright, 1999; Hiei et al., 1997; Hu et al., 2003; Shibata and Liu, 2000). There are currently few published data

Table 1 Summary of current published data on A. tumefaciens transformation giving fertile adult wheat plants. Wheat cultivar Bobwhite Fielder Florida Cadenza Veery-5 Bobwhite Bobwhite Bobwhite Spring winter S S W S/W S S S S Explant PCIE, EC PCIE IE IE EC PCIE EC PCIE EC PCIE Agrobacterium strain (vector) C58 (pMON18365) AGL0 (pBGX1) AGL1 (pAL154/ 156) AGL1 (pAL154/ 156) LBA4404 (pHK21) C58 (pMON18365) C58 (pMON18365) C58 (pPTN155) Transformation freq. (%) 1.44.3 1.8 0.33.3 2.5 1.23.9 4.4 4.819 0.51.5 No. of plants reported O100 4 39 5 Khanna and Daggard (2003) 3354 154 Haliloglu and Baenziger (2003) References Cheng et al. (1997) Weir et al. (2001) Wu et al. (2003) Wu et al. (2003) Khanna and Daggard (2003) Hu et al. (2003) Cheng et al. (2003) Haliloglu and Baenziger (2003)

PCIE, pre-cultured immature embryos; EC, embryogenic cultures. IE, fresh immature embryos.

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specically for wheat but the little there are support these advantages. Cheng et al. (1997) compared wheat transformed by either particle bombardment or by Agrobacterium; only 17% (13/77) of the bombarded lines contained a single transgene copy compared with 35% (9/26) of those transformed by Agrobacterium. Another study from the same company concluded that 56% (40/77) of the bombarded lines showed a 3:1 segregation ratio indicative of a single locus integration compared with 47% (45/97) for Agrobacterium transformation, but also showed that more of the Agrobacterium lines contained a single transgene copy (4 out of 6 tested compared with 1 out of 5 for the biolistics lines) (Hu et al., 2003). Data from our laboratory (Wu et al., 2003) support these ndings with 22/44 Agrobacterium lines segregating at 3:1 and 12/20 with single copies. 2.4. Tissue culture and selection In common with other cereals, the principal route to recover plants from transformed somatic wheat cells is via somatic embryogenesis. Somatic cells with good embryogenic potential are ideal targets for chromosomal integration of transgenes since each has the potential to become an individual plant. Somatic embryos originating from callus derived from immature scutella or immature inorescences can be induced with phytohormones to germinate to form shoot and root structures and give rise to fertile adult plants. The basic salt formulation used in most wheat tissue culture protocols is solidied MS (Murashige and Skoog, 1962) supplemented with vitamins, hormones and sugars. The auxin 2,4-D (2,4-dichlorophenoxyacetic acid) is frequently used to induce embryogenesis, although the use of picloram has also been described (Barro et al., 1998; Campbell et al., 2000), either with or without low levels of cytokinins such as zeatin and benzyladenine purine (BAP). In shoot regeneration, higher levels of cytokinins are employed either with or without lower auxin levels. Osmotic treatment is thought to offer protection to bombarded material by minimising cytoplasm leakage from target cells (Vain et al., 1993). Various osmotic treatments, induced by incubation on media containing different combinations of sugar-alcohols such as mannitol and sorbitol, have been employed prior to bombardment (Altpeter et al., 1996; Blechl and Anderson, 1996; BrinchPedersen et al., 2000; Jordan, 2000; Ortiz et al., 1996; Stoger et al., 1999a; Weeks et al., 2000). A rafnose/ mannitol combination has also been successfully employed (Campbell et al., 2000). One of the sugars most commonly included is sucrose, often at 23% concentration (Chen et al., 1998; Deblock et al., 1987; Nehra et al., 1994; Witrzens et al., 1998) and the effect of a range of sucrose concentrations has been investigated (Rasco-Gaunt et al., 2001). An additional approach for osmotic shock, involving a temporary transfer of material from 2% maltose to 20% maltose, has also been used (Uze et al., 1999).

Although commonly used in biolistic transformations, osmotic treatments do not have the same positive effects on T-DNA delivery in wheat (Cheng et al., 2003). However, immature wheat embryos or embryogenic callus that are subjected to brief periods of desiccation do show greatly enhanced T-DNA delivery and plant tissue recovery after Agrobacterium co-cultivation (Cheng et al., 2003). The length of pre-culture of explant material is usually balanced against medium composition to optimise the stage of cellular differentiation, cell cycle and osmotic conditioning. The relatively low rates of transformation necessitate the use of chemical selection systems to kill or compromise the growth of untransformed tissues and allow the preferential survival of transformed plants. Regimes based on negative and positive selection are routinely used for wheat and comprise two components; a selection agent added to the tissue culture medium, such as an antibiotic, herbicide or a particular carbon source and the incorporation into the transformation cassette of a gene conferring selective advantage under the specic media conditions. Commonly used selection agents for wheat are those based on formulations containing the herbicide phosphinothricin (PPT), and those based on aminoglycoside antibiotics (Goodwin et al., 2004). PPT acts by irreversibly inhibiting glutamine synthetase, a key enzyme for ammonium assimilation and nitrogen metabolism in plants (Deblock et al., 1987) resulting in increasing levels of ammonia which is toxic to the cells (Tachibana et al., 1986). Two genes isolated from different soil microorganisms, bar (Basta resistance) (Murakami et al., 1986; Thompson et al., 1987) and pat (Wohlleben et al., 1988), encode the enzyme phosphinothricin acetyl transferase (PAT) which converts PPT to the non-toxic acetylated form and allows the growth of transformed cells in the presence of PPT or commercial glufosinate ammoniumbased formulations such as Basta and Challenge. Glufosinate ammonium also forms the basis of the LibertyLink range of herbicide-tolerant crops. Another commonly applied selection system for wheat uses aminoglycoside antibiotics such as kanamycin, neomycin, gentamycin, G418 and hygromycin which inhibit protein synthesis. These antibiotics can be inactivated by phosphotransferases encoded by various genes including nptII (neo) (Bevan et al., 1983) and hpt (aphIV) (Vandenelzen et al., 1985; Waldron et al., 1985). Kanamycin selection became a laboratory standard for many dicotyledonous species but the cereals and other grasses demonstrated innate tolerance to relatively high levels of this antibiotic (Hauptmann et al., 1988). Instead, hygromycin and G418 proved useful alternatives for maize, wheat, barley, rice, and the meadow grass Lolium (Barcelo et al., 2001; Wilmink and Dons, 1993). Driven partly by perceived risks of horizontal and vertical gene transfer, a range of more environmentally benign selection systems have been developed. The most advanced of these is the phosphomannose isomerase (PMI)

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system (Syngenta) which utilises the manA gene to convert the predominant carbon source, mannose-6-phosphate, to fructose-6-phosphate for respiration (Joersbo, 2001; Joersbo et al., 1999). Not only are the untransformed cells deprived of a carbon source, but the unused mannose-6-phosphate accumulates and has additional negative effects including inhibition of glycolysis, possibly due to phosphate starvation. PMI selection has been shown to be superior to antibiotic and herbicide selection for wheat (Reed et al., 2001). 2.5. Chimaerism and somaclonal variation Chimaerism and somaclonal variation are generally undesirable in transgenic plants used to study specic genes. Chimaerism is caused when the cells comprising a somatic embryo are heterogeneous for the same transgenic event. Although tissue culture and selection conditions are manipulated to encourage only whole plants that are derived from one original transformed cell to regenerate, the transformation may have occurred in a cell or cells that were derived from the original progenitor cell and from which subsequent divisions lead to an unequal distribution of transformed and non-transformed sectors within the same plant. Such plants are called chimaeras and depending upon whether transgenic sectors lead to gametic tissue development, some ears from these plants may not contain any transgenic seeds. Chimaerism is one cause of poor or zero transgene inheritance to the T1 generation. Somaclonal variation is the term used to describe the genetic instability often induced by biotic and abiotic stresses including as a consequence of cell/tissue culture. Tissue culture-induced somaclonal variation has been widely exploited as a source of variation for breeding material resulting in commercially available varieties (for example potato). It has also been used experimentally in wheat to generate novel sources of herbicide tolerance (Bozorgipour and Snape, 1997) and disease resistance (Mehta and Angra, 2000) and to look for variation in high molecular weight glutenin subunit genes (Beshkova et al., 1998). The chances of creating unintentional somaclonal variants as part of the transformation and tissue culture processes by reducing to a minimum known causative factors such as long culture periods and high levels of auxin. However, as it is unlikely that all tissue culture-induced somaclonal variation can be totally eliminated, strategies should be adopted to identify and remove variants from the population prior to analysis. One practical way to deal with these relatively rare and random events in a population of independent transgenic lines is to eliminate individuals possessing gene expression proles and phenotypes that are not shared by other transgenic events in the same population.

3. Targeting transgene expression For some applications, high level constitutive transgene expression in all tissues at all stages of development is required. However for others, the expression of a transgene can be effectively targeted to specic tissues or to particular stages of plant development by including in the transformation cassette, promoters that give the desired pattern of regulation. The ability to make these chimaeric expression cassettes that combine a promoter sequence from one gene with the coding sequence from another, forms an important level of experimental design. Clearly the choice of promoter depends on the particular research objective and trait under investigation. For applications involving food processing or nutritional quality, the principal target tissues are the endosperm, embryo and aleurone. A list of promoters reported to be active in these tissues from experiments with stably transformed wheat is shown in Table 2. The cauliower mosaic virus (CaMV) 35S promoter is one of the most commonly used in dicots but has poor activity and a tendency to be silenced in wheat unless enhancer elements are incorporated (Chen et al., 1998, 1999). The promoters that appear to give the highest and most stable constitutive expression in wheat are the maize ubiquitin promoter and intron (Ubi1) (Christensen et al., 1992) and the rice actin promoter including the 5 0 intron (Act1) (McElroy et al., 1991). Although the Ubi1 promoter is considered constitutive, recent studies to characterise its specicity in detail have shown that position effects, developmental stage (Rooke et al., 2000) and stress (Stoger et al., 1999b) may all affect its activity in transgenic wheat lines. The promoter from the recently identied maize histone H2B gene also gives strong, constitutive expression in wheat when cloned upstream of the Ubi rst intron (Rasco-Gaunt et al., 2003). There is currently a lack of well characterised wheatderived promoters and promoters from other species are frequently employed in wheat transgenics. However, such heterologous promoters do not always maintain the activity originally observed in the species of origin. For example, the promoter from the wheat Glu-1D-1 high molecular weight subunit (1Dx5) gene (from K1191 to transcription start codon) has been shown to be tightly endospermspecic when directing the expression of the UidA (GUS) gene in transgenic durum wheat (Lamacchia et al., 2001). However, in addition to expression in the endosperm, this promoter has been observed to also drive GUS expression in roots, leaves and pollen of transgenic barley (Zhang, 2001) and in the glumes and aleurone of transgenic oats (Perret et al., 2003). The wheat 1Dx5 promoter driving the genomic Glu-1Dx5 fragment in transgenic maize was reported to retain its endosperm-specicity but was inherited inefciently through the male germ-line implying there was some pollen abnormality (Sangtong et al., 2002). The promoter of the wheat ADPglucose pyrophosphorylase gene (a wheat homologue of the maize shrunken2 gene encoding an

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Table 2 Promoters active in seed tissues from stably transformed wheat Promoter Ubiquitin (Uib1) Actin (Act1) 35S 35S-Sh ex/int 35S-35S 35S-adh1 35S-RTBV adh1/pEmu Histone H2B Alcohol dehydrogenase Rice trungro bacilliform virus Granule-bound starch synthase HMWG subunit 1Dy10 Low molecular weight glutenin (LMWG1D1) HMWG subunit (1Bx17) HMWG subunits Glu-D1-1b (1Dx5) and A1-1a (1Ax1) ADPglucose pyrophosphorylase Globulin Glb1 Amylase (Amy1 and Amy2 Puoindolines a and b Lipid transfer protein (Ltp) General activity Constitutive Constitutive Constitutive Constitutive Constitutive Constitutive Modications Including rst intron Including actin intron Maize shrunken exon and intron Maize adh1 intron Rice tungro bacilliform virus intron Maize AREs and Adh1 intron. Octopine synthase Ubi rst intron Source Rooke et al., (2000) Rice CaMV CaMV CaMV CaMV CaMV Maize Maize Maize RTV Wheat Blechl and Anderson (1996) Wheat Wheat Genomic clones Wheat Example reference Rooke et al., (2000) Jordan (2000) Chen et al. (1999) Karunaratne et al. (1996) Zhou et al. (1995) Barro et al. (1998) Bieri et al., (2003) Karunaratne et al. (1996) Rasco-Gaunt et al. (2003) Vasil et al. (1992) Sparks and Jones (unpublished) Kluth et al. (2002) Blechl and Anderson (1996) Stoger et al. (1999a) Sparks, Tamas and Jones (unpublished) He et al. (1999); Lamacchia et al., (2001) Thorneycroft et al. (2003) Sparks and Jones (unpublished) Stone (2003) Wiley, Shewry and Jones (unpublished) Simmonds et al. (1998)

Constitutive Constitutive Constitutive Phloem/constitutive Endosperm and pericarp Endosperm Endosperm Endosperm Endosperm

Endosperm, aleurone and sub aleurone Endosperm Aleurone/scutellum Endosperm Aleurone

Genomic clone plus deletion series K1391

Wheat Maize Wheat Wheat Wheat

enzyme of starch synthesis) has been shown to drive strong expression in embryos and guard cells of transgenic tobacco and in the endosperm and aleurone of transgenic wheat plants starting at ve days after owering (Thorneycroft et al., 2003). With the aim of compiling a catalogue of suitable promoters for targeting wheat endosperm, expressionprole analysis of transgenic wheat lines expressing the UidA gene under the control of a range of promoters including a maize globulin 1 promoter, the 1Dx5 and Bx17 HMW glutenin promoters and the rice tungro bacilliform virus promoter is underway (see Fig. 1) (Jones et al., 2003).

4. Genetic approaches to the study of grain development and composition Good bread making quality is a key goal for wheat breeding. It is mainly determined by dough strength as a result of the interactions between gluten proteins. Of the many proteins that make up gluten, one type, the high molecular subunits of glutenin (HMWG) are particularly

amenable to modication by transformation (Shewry et al., 1995) and most of the published work to date that used a transgenic approach to modify grain composition has focussed on these proteins. For example, Blechl and Anderson (1996) demonstrated that a hybrid Dy10/Dx5 HMWG subunit protein was over-expressed in the model wheat cultivar Bobwhite and accumulated to levels similar to native HMWG subunits. The same year, Altpeter et al. (1996) reported that a genomic 1Ax1 clone, also transgenically introduced into Bobwhite, resulting in a marked increase in total HMWG subunit protein. Mixograph studies on dough from transgenic lines expressing genes encoding 1Ax1 and 1Ax1 plus 1Dx5 (Anderson et al., 1989; Halford et al., 1992) showed a step-wise increase in both the peak dough resistance and the mixing time (Barro et al., 1997). In one particular transgenic line, highly overexpressing the 1Dx5 transgene, the proportion of HMWG subunit proteins in the gluten fraction was increased from 12% to over 20% with the dough proving too strong for conventional bread making (Rooke et al., 1999). The lines described by Barro et al. (1997) were also grown in replicate eld trials and subjected to small-scale milling and baking tests which

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Fig. 1. Patterns of GUS staining in wheat plants transformed with the UidA gene under the control of various promoters: (a) the maize ubiquitin (Ubi1) promoter in immature inorescences; (b) 1Dx5 promoter in immature seeds; (c) rice tungro bacilliform virus promoter in mature leaves; (d) rice actin (Act1) promoter in carpel and stamens (Photographs courtesy of Caroline Sparks, Rothamsted Research).

revealed changes in elastic modulus of the gel protein fraction, loaf volume and crumb structure between transgenic lines and controls (Darlington et al., 2003). Other strategies for research into grain development and composition include the engineering of gross or ne changes to the amino acid composition. In one such approach to investigating the role of large repetitive domains within the 1Dx5 HMWGHMWG protein, He et al. (2000) attempted to create a series of lines transformed with modied 1Dx5 subunit genes engineered with repetitive domains about 34 and 17% shorter, and 22% larger than the native 1Dx5 protein (DOvidio et al., 1997). One of the lines generated, which expressed the larger 1Dx5 form, is currently undergoing analysis to determine the impact of the introduced gene on functional properties. One potential drawback of the transgenic approach is the difculty of distinguishing between native proteins and

protein encoded by the transgene. This is particularly acute when dealing with classes of highly abundant and homologous storage proteins such as the HMWG subunits. Recent work in our laboratory has involved engineering amino acid substitutions in low molecular weight glutenin subunit proteins to study the role of specic cysteine residues and to overcome the problem of protein recognition by utilising an epitope tag to follow protein trafcking and accumulation. The incorporation of the c-myc epitope tag at the 3 0 end of the coding region did not compromise correct folding or incorporation of low molecular weight subunits into gluten polymers and allowed expression and protein trafcking to be unambiguously followed (Tosi et al., 2000, 2004). The success of transgenic approaches to modication of wheat HMWG subunit proteins suggests that it may be possible to target starch, another major seed storage

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compound. Transgenic approaches to alter expression levels of starch biosynthetic genes have been particularly successful in potato and maize (for recent reviews see Bligh, 1999; Rahman et al., 2000; Rossi et al., 2001; Slattery et al., 2000) and a search of patent databases reveals signicant industrial interest in genetic modication of starch quality in wheat. Potential applications include wheat starches that mimic starch mutants (e.g. waxy) in maize, phosphorylated starch (currently obtained from potato) and thermoplastic and biodegradable starches for packaging. Target genes are those that encode the various isoforms and classes of soluble and granule-bound starch synthases, which could be manipulated to ne-tune the proportions of amylose and amylopectin, and produce designer starches with novel functional properties (Mullerrober and Kossmann, 1994). Although there has been signicant research activity in industrial laboratories, little is in the public domain. The only published report utilised the shrunken2 gene from maize, encoding the large subunit of ADP-glucose pyrophosphorylase, which plays a key role in the regulation of starch biosynthesis in cereals. This gene was overexpressed in wheat leading to higher seed yield and larger biomass in plants grown under glasshouse conditions (Smidansky et al., 2002). An RNAi-mediated approach to downregulate granule-bound starch synthase (GBSS) in transgenic wheat has been successful in making a partial waxy starch containing 18% amylose compared with 30% in the null controls (unpublished data of Crouch, Day, Lazzeri and Jones).

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Acknowledgements Rothamsted Research receives grant aided support from the Biotechnology and Biological Sciences Research Council UK.

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