Correlations of the Amount of Gluten Protein Types to the Technological Properties of Wheat Flours Determined on a Micro-scale

Flour samples of 14 wheat cultivars previously characterised by rheological measurements and by baking tests on a micro-scale (Kieffer et al.: Journal of Cereal Science27 (1998) 53–60) were analysed for the relative amounts of gluten protein types using a combined extraction/HPLC procedure. Regression analysis was used to find relations between wheat properties and protein quantities. The results indicated that the maximum resistance of dough and gluten and the gluten index were strongly dependent on the quantity of glutenin subunits (GS) in flour; additionally they were influenced by the ratio of gliadin to glutenin subunits. Within the family of glutenin proteins, the correlation coefficients for high-molecular-weight (HMW) and low-molecular-weight (LMW) GS were in a similar range, but twice the amount of LMW GS was necessary to get the same resistance as with HMW GS. Among HMW GS, the contribution of x-type GS was more important than those of y-type GS. The extensibility of dough and gluten was mainly dependent on the ratio of gliadin to total glutenin subunits, to HMW GS and LMW GS. Dough development time showed the highest correlation with total HMW GS and x-type HMW GS. Bread volume was influenced by the total amount of gluten protein more than by the amount of protein in different groups or of different types, probably because of the rather low range of flour protein content (8·7–12·0 %) within the set studied. Significant differences between gliadins and glutenins with respect to their effects on bread volume could not be detected. The correlation between bread volumes and the quantity of gluten proteins was higher, when dough was mixed to optimum.     

Distribution and function of LMW glutenins,HMW glutenins,and gliadins in wheat doughs analyzed with ‘in situ’ detection and quantitative imaging techniques

The different gluten subunits, gliadins, LMW glutenins, and HMW glutenins have been reported to play different key roles in different type of wheat products. This paper studied the interaction between gliadin, LMW and HMW glutenins in soft, hard and durum semolina flour doughs during different stages of mixing. In order to see how do the gluten subunits (gliadin, LMW glutenin and HMW glutenin) redistribute during mixing, dough samples were taken at maximum strength and 10 min after maximum strength. The doughs have been mixed with the same level of added water (55%), therefore they all have different strengths values due to their changes in proteins content. Oscillatory rheological measurements were performed on the doughs. It has been found that HMW glutenins are relatively immobile because of their less molecular mobility and do no redistribute themselves especially at high strength for doughs such as hard wheat flour. LMW glutenins and gliadins on the other hand redistribute themselves at even at high dough strengths forming a more stable network. In weaker doughs such as soft wheat, the breakdown of the three proteins subunits is responsible for the decay in dough strength. We have also visualized how the greater amount of LMW glutenins in semolina is in constant interaction with HMW glutenins and gliadins allowing the dough to maintain a stable strength for an extended mixing time. Finally, we have found the ‘in situ’ detection and quantitative analysis techniques to be more sensitive to the changes occurring in the gluten network of the dough than the oscillatory rheological analysis.     

小麦-卵穗山羊草1Mg异附加系蛋白质品质形成解析

为了突破小麦品质育种瓶颈,从小麦的近缘属种中挖掘优质蛋白亚基,并解析优质亚基的品质效应,本研究选取中国春(CS)及CS与卵穗山羊草1Mg异附加系(CS-1Mg)作为试验材料,研究其亚基组成、籽粒发育过程中的谷蛋白聚合体动态积累、面筋蛋白的微观结构与二级结构、面团的混揉特性等以解析异源染色体的加入对面团品质形成的影响。结果表明,与CS相比,CS-1Mg中出现了新型HMW-GS,使其在籽粒发育过程中UPP快速积累,面筋蛋白呈现出更好的二级结构分布,微观结构上蛋白质交织更紧密,流变学特性及混揉特性增强,品质显著提高。CS-1Mg所携带的新型HMW-GS可以作为优质基因源用于优质小麦育种,CS-1Mg可作为中间材料应用于小麦品质改良的生产实践中。     

小麦高、低分子量麦谷蛋白亚基对品质性状的影响

为给小麦品质改良提供理论依据,以6个国家的532个品种(系)为材料,分析了小麦高、低分子量麦谷蛋白亚基与品质性状的关系。结果表明,各位点上不同的等位变异对品质性状的效应存在显著差异,亚基1、2*、17+18、5+10、GluA3f、GluB3b和GluB3g对面团形成时间和稳定时间具有显著的正向效应;亚基N、1、14+15、17+18、7+8、2+12、3+12、4+12、GluB3d、GluB3f、GluB3h和GluB3g对蛋白质和湿面筋含量具有显著正向效应;从亚基对品质性状的综合影响来看,1、17+18、GluA3f、GluB3g和GluB3f可作为优势亚基。具有1、17+18、2+12和1、17+18、5+10高分子量麦谷蛋白亚基组合的品种(系)综合品质性状显著优于含有N、14+15、2+12亚基组合的品种(系);具有低分子量麦谷蛋白亚基组合GluA3c、GluB3g,GluA3d、GluB3g和GluA3f、GluB3b品种(系)的综合品质性状显著优于含有GluA3b、GluB3j亚基组合的品种(系)。     

Durum wheat breadmaking quality: Effects of gluten strength,protein composition,semolina particle size and fermentation time

The effects of particle size of granulars (semolina and flour combined), gluten strength, protein composition and fermentation time on the breadmaking performance were compared for eleven durum wheat genotypes of diverse strength from North America and Italy grown in the same environment. All genotypes were γ-gliadin 45 types (low-molecular weight glutenin subunit 2 patterns) associated with superior pasta-making quality. Three cultivars with high-molecular weight glutenin subunit 20 exhibited relatively weak gluten, confirming that this subunit is associated with weakness in durum wheat. Gluten strength as measured by a range of technological tests was directly and strongly related to the proportion of insoluble glutenin (IG) in granulars protein as determined by a spectrophotometric procedure. Reducing the particle size of granulars by gradual reduction shortened development time in both the farinograph and mixograph. Reducing granulars also increased starch damage and, accordingly, farinograph water absorption, but remix-to-peak baking absorption was unaffected due to increased fermentation loss for finer granulars. Neither loaf volume, nor remix-to-peak mixing time were affected by the particle size of the granulars indicating that regrinding is not an asset for baking provided there is adequate gassing power. Loaf volume was directly related to gluten strength and IG content, and inversely related to residue protein, a non-gluten containing fraction. When fermentation time was reduced from the standard 165 to 90 min and 15 min, all genotypes exhibited a progressive increase in loaf volume. Therefore, regardless of strength, short fermentation time is preferred when high volume durum wheat bread is desired. Some of the stronger durum genotypes exhibited remix-to-peak bread volume comparable to that expected of good quality bread wheat, indicating that there is potential to select for genotypes with improved baking quality in conventional breeding programs by screening for high content of insoluble glutenin.     

Baking quality of emmer-derived durum wheat breeding lines

Durum wheat (Triticum turgidum L. var. durum) is used predominantly for pasta products, but there is increasing interest in using durum for bread-making. The goal of this study was to assess the bread-making potential of 97Emmer19, an Emmer wheat (Triticum turgidum L. var. dicoccum) and in breeding lines derived from crosses of 97Emmer19 with adapted durum wheat cultivars. 97Emmer19 and its progeny were evaluated in 2005 and 2006 along with five durum wheat cultivars. Three bread wheat (Triticum aestivum L.) cultivars were included as checks to provide a baseline of bread making quality observed in high quality bread wheat cultivars. 97Emmer19 exhibited higher LV than all the durum wheat checks and approached the LV achieved with the bread wheat cultivar ‘AC Superb’. Breeding lines derived from 97Emmer19 had higher LV than those of the durum wheat checks, confirming that this trait was heritable. In general, durum wheat cultivars with elevated gluten strength and/or increased dough extensibility were noted to have higher LV. Dough extensibility appeared to be a more critical factor as gluten strength increased. These results indicate that there is potential to select for genotypes with improved baking quality in durum breeding programs.     

Elastic properties of gluten representing different wheat classes

Traditional instruments used to evaluate dough and/or gluten rheological properties do not provide unambiguous separation of elastic and viscous behaviors. Recovery after shear creep and cyclic large deformation cyclic tensile testing were used here to decouple elastic and viscous effects. A large variation in the recoverable shear strain (∼7.2% to ∼28%) was seen for glutens from 15 U.S. popular common wheat cultivars with varying HMW subunits. Sedimentation values ranged from 29 to 57 ml for 12 hard wheat cultivars and 15 to 22 ml for three soft wheat cultivars. The tensile force at 500% extension ranged from 0.12 to 0.67 N for hard wheat glutens and from 0.10 to 0.20 for soft wheat glutens. However, the recoverable work after large extension was less than 40% of the total work of extension. In addition, recoverable work in tensile testing was highly correlated with the total work of extension (r² = 0.97) and mixograph mix times (r² = 0.81). Good to excellent bread volume was obtained for several cultivars from this sample set. This suggests that optimizing water absorption for mixing doughs to achieve maximal bread volume compensates for the wide range of viscoelastic behaviors of gluten.     

安徽省小麦产业体系展示品种HMW-GS组分及GMP的研究

为了解安徽省淮北及黄淮麦区的小麦加工品质,以52个安徽省小麦产业体系展示品种为材料,分析了供试小麦品种HMW-GS的组成和分布、谷蛋白聚合体(GMP)含量、小麦蛋白质含量、Zeleny沉降值及湿面筋含量。结果表明,52个供试小麦品种的HMW-GS变异类型众多,共发现13种变异类型,包含6种优质亚基类型;共出现了21种HMW-GS组合。按照GMP、蛋白质、湿面筋含量及Zeleny沉降值可将52个供试品种分为4类。供试品种的品质评分介于4~10分之间,平均7.4分。基因位点Glu-A1含有1亚基的品种,GMP含量高于含有Null亚基的品种。     

Incorporation of high-molecular-weight glutenin subunits into doughs using 2 gram mixograph and extensigraphs

To study the contributions of high-molecular-weight glutenin subunits (HMW-GS) to the gluten macropolymer and dough properties, wheat HMW-GS (x- and y-types) are synthesized in a bacterial expression system. These subunits are then purified and used to supplement dough mixing and extensigraph experiments through dough partial reduction and reoxidation to allow these exogenously added HMW-GS to incorporate into gluten polymers. Detailed results are given for seven mixing and two extension parameters. HMW-GS synthesized in bacteria behaved similarly under these conditions to the same HMW-GS extracted from wheat flour. These experiments initially focused on the HMW-GS of the D-genome of hexaploid wheat encoded at the Glu-D1 locus; e.g. the Dx2, Dx5, Dy10, and Dy12 subunits. Experiments used five different flours and results are shown to be consistent when normalized to results from Dx5. The incorporation of Dx-type subunits into the gluten disulfide bonded network has greater effects on dough parameters than incorporation of Dy-type subunits. When Glu-D1 x- and y-type subunits are incorporated together, there are synergistic effects greater than those with either subunit type alone. This synergistic effect was greatest with approximately equal amounts of Dx- and Dy-type subunits - implying a 1:1 stoichiometric relationship.     

长江中下游麦区小麦品质改良设想

长江中下游麦区是我国唯一的弱筋小麦优势产业带,该麦区的沿江、沿海地区适宜发展弱筋小麦,其他地区适宜发展中筋小麦.弱筋、中筋小麦品种的品质遗传改良是该麦区重要育种方向之一.目前该麦区虽然育成并推广了一批弱筋、中筋小麦品种,基本满足了生产上对优质小麦品种的需求,但还存在一定的问题.如新育成的弱筋小麦品质并不优于扬麦9号和宁麦9号,中筋小麦面筋含量和强度还需提高.针对存在的问题,提出该麦区小麦品质改良的3个设想:(1)利用高分子量谷蛋白亚基缺失,降低面筋强度,培育优质弱筋小麦;(2)利用优质高分子量麦谷蛋白亚基组合,改善蛋白质组分,提高面筋强度,培育优质中筋小麦;(3)利用Wx蛋白亚基缺失,培育弱筋和中筋兼用型小麦,使该麦区小麦品种在蛋白质含量和面筋含量偏高而不能作为弱筋小麦使用时,利用其优良的淀粉品质加工品质较优的面条.     

Effect of incorporation of an i-type low-molecular-weight glutenin subunit and a modified γ-gliadin in durum and in bread wheat doughs as measured by micro-mixographic analyses

The effects of incorporation of an i-type low-molecular-weight glutenin subunit (LMW-i) and of a modified γ-gliadin showing an additional cysteine residue, on 2 g Mixograph parameters of durum (biotypes 42 and 45 of the Italian cv. Lira) and bread wheat (Australian cv. Kukri) doughs were studied. In bread wheat flour incorporation of the modified γ-gliadin resulted in a significant decrease in dough strength (decreased mixing time and peak resistance), but at the same time it produced a slight increase in dough stability (decreased resistance to breakdown). The incorporation of the LMW-i type into bread wheat dough had minimal effects on dough mixing requirements. The incorporation of both LMW-i type and modified γ-gliadin in durum wheat doughs produced a significant decrease in the overall dough strength, especially in Lira 45 biotype doughs. Reversed phase high-performance liquid chromatography (RP-HPLC), size exclusion high-performance liquid chromatography (SE-HPLC) and two-dimensional gels analyses of control and reconstituted semolina doughs showed that the two polypeptides were in the polymeric fraction. The effect of the incorporation of the two polypeptides in durum and bread wheat doughs showed remarkable differences and the reasons for this is discussed in terms of both intrinsic differences between wheat flour and durum semolina and in methodological approaches.     

Gluten proteome comparison among durum wheat genotypes with different release date

In order to deepen insight into durum wheat prolamin composition in relation to both end use quality and gluten related disorders, a phenotyping of four genotypes of different release date and technological performance was carried out. A proteomic approach integrated with the evaluation of protein aggregation level, amino acid composition and reactivity to G12 monoclonal antibody, was adopted. The degree of polymerization, estimated as unextractable polymeric proteins (UPP), was positively influenced by Cys-rich proteins (Glu-B3 LMW-GS), with Saragolla showing up to 40% higher values than other genotypes. The proteomic assessment allowed to identify proteins involved in gluten related disorders. In particular, ω5-gliadin involved in wheat allergy (WA), resulted markedly over-expressed in the old landrace Dauno III, up to four-fold than in modern Saragolla. A marked influence of genotype on the expression level of gliadins containing toxic epitopes (TECP) was observed with the more recent genotypes showing lower values (−53%). Also, reactivity to G12 moAb resulted higher in the two old genotypes consistently to their higher celiac disease (CD) toxicity evaluated by the proteomic approach. In conclusion a better gluten composition for processing seems to be associated to a lower expression level of CD toxic peptides and Tri a 19.     

Composition of polymeric proteins and bread-making quality of wheat lines with allelic HMW-GS differing in number of cysteines

Three lines of Australian wheat variety Avocet, two biotypes of Australian variety Halberd, and the Italian bread wheat cultivar Fiorello and its derived line were used to study the possible role of the Glu-B1 HMW-GS in polymeric protein composition and flour functionality. These sets of lines contain HMW-GS 7+8, 7+9, 20x+20y or 26+27. Subunit Bx7 has four cysteine residues, while subunit Bx20 has only two. Compared to Avocet A (7+8), line Avocet C (20x+20y) exhibited an increase in polymeric protein, a decrease in the gliadin-to-glutenin ratio, and a marked decrease in unextractable polymeric protein (UPP). Avocet C doughs exhibited greater extensibility and shorter Mixograph dough development times, and baked into smaller loaves compared to those from Avocet A. Similar differences were observed in comparisons between the two Fiorello lines and between the two Halberd biotypes that had differing Glu-B1 alleles. These results show that in closely related genotypes, the presence of the Bx7+By8 or Bx7+By9 HMW-GS instead of Bx20+By20 or Bx26+By27 is associated with higher UPP, higher dough strength and better bread-making performance. It is suggested that this could be due to the greater number of cysteines in the Bx7 subunit that are available for forming higher-molecular-weight glutenin polymer. This could give the possibility to manipulate dough properties and bread-making quality by utilizing HMW-GS alleles with varying numbers of cysteine residues.     

Impact of late-season N fertilisation strategies on the gluten content and composition of high protein wheat grown under humid Mediterranean conditions

The rise in high protein common wheat in humid Mediterranean areas has determined a need to compare specific and effective nitrogen (N) fertilisation protocols in order to increase their end-use value. The aim of the work was to assess the impact of late-season N fertilisation strategies on grain yield and protein content (GPC), gluten fraction composition, and rheological traits. Different applications and types of fertiliser (soil applied ammonium nitrate, soil applied urea, foliar applied urea and a foliar applied commercial fertiliser) were distributed at the same rate (30 kg N ha⁻¹) in a field experiment in NW Italy, during three growing seasons. A control without any late-season N fertilisation was also considered. All the treatments received 130 kg N ha⁻¹ as ammonium nitrate (AN), which was split between tillering and the beginning of the stem elongation growth stages.None of the compared late-season N fertilisations significantly affected canopy greenness and stay green duration during the grain filling period, or the grain yield, test weight, and thousand kernel weight, although the foliar application significantly increased foliage burning (+9.8%). The late application of N consistently increased GPC (+1.1%) and dough strength (W, +21%) in the different growing seasons. The type of fertilisation strategies clearly affected the gluten content and rheological parameters: AN was more effective than urea as a soil top-dressed applied fertiliser in increasing W (+10%), as a result of a higher rise in the GPC content (+0.5%) and extensibility (L, +11%). The foliar application at anthesis, at the same N rate, led to a comparable GPC and W with those of the soil top-dressed granular fertiliser. Only a weak effect of granular urea on y/x type HMW was observed for the gluten composition. Conversely, a notable influence of year was observed (i.e. GS/Glia and y/x type HMW), which in turn resulted in a significant impact on W and P and on the aggregation time and aggregation energy.This study offers a further contribution to the improvement of specific N fertilisation strategies in order to enhance the wheat quality according to its end-use value.     

HMW-GS标记辅助选育优质小麦新品系

为加速优质小麦育种进程,将聚合杂交、HMW-GS辅助选择与常规育种程序结合,在小麦品种烟农19、济麦20、郑优6号、百农66不同杂交组合F2代进行HMW-GS标记辅助选择,经连续多代鉴定筛选,选育出14个含优质HMW-GS或HMW-GS组合的小麦新品系.鉴定结果表明,14个新品系的蛋白质含量、沉降值、湿面筋含量、面筋指...     

Bread making quality of durum wheat genotypes with some novel glutenin subunit compositions

Durum wheat genotypes with some novel high M_r (high molecular weight, HMW) and low M_r (low molecular weight, LMW) glutenin subunits were grown in Sicily for two years of testing in order to compare their rheological and baking properties with respect to commercial durum wheat cultivars. Good bread making quality, as measured by Alveograph W and P/L, Farinograph and Mixograph parameters, and loaf volume was observed in genotypes combining high M_r subunits 2⁺, 1 or 11 encoded at the Glu-A1 locus with the so-called LMW-2 subunit group encoded at the Glu-B3 locus. The cultivar Avanzi, which carries high M_r subunit 2⁺ and LMW-2-like subunits, and the cultivars Dritto and Keops, which contain novel high and low M_r subunits, gave higher loaf volumes than control cultivars. The LMW-2 group subunits were found to be the main factor in determining dough strength (Alveograph W). The increase in the amount of high M_r subunits in genotypes with one expressed Glu-A1 gene may account for their improved rheological and baking properties.     

Large deformation stress relaxation and compression-recovery of gluten representing different wheat classes

Despite the great variety of physicochemical and rheological tests available for measuring wheat flour, dough and gluten quality, the US wheat marketing system still relies primarily on wheat kernel hardness and growing season to categorize cultivars. To better understand and differentiate wheat cultivars of the same class, the tensile strength, and stress relaxation behavior of gluten from 15 wheat cultivars was measured and compared to other available physicochemical parameters, including but not limited to protein content, glutenin macropolymer content (GMP) and bread loaf volume. In addition, a novel gluten compression–relaxation (Gluten CORE) instrument was used to measure the degree of elastic recovery of gluten for 15 common US wheat cultivars. Gluten strength ranged from 0.04 to 0.43 N at 500% extension, while the degree of recovery ranged from 5 to 78%. Measuring gluten strength clearly differentiated cultivars within a wheat class; nonetheless it was not a good predictor of baking quality on its own in terms of bread volume. Gluten strength was highly correlated with mixograph mixing times (r = 0.879) and degree of recovery (r = 0.855), suggesting that dough development time was influenced by gluten strength and that the CORE instrument was a suitable alternative to tensile testing, since it is less time intensive and less laborious to use.     

小麦HMW GS检测方法的优化及应用

为了准确快速鉴定小麦品种的HMW-GS组成,以18个已知HMW-GS组成的品种为对照,将4种不同浓度分离胶的SDS-PAGE与分子标记(Bx7、Bx7OE和By 8)相结合,构建一套适于检测HMW-GS组成的方法,并用该方法检测214份陕西小麦品种(系)的HMW-GS组成。4种分离胶浓度中,除了亚基7、7*与7OE和8与8*外,9%的分离胶可以很清楚地分离Glu1-位点的其他常见亚基,结合分子标记(Bx7、Bx7OE和By 8)检测对照品种,结果与已知亚基(基因)一致。用分离胶浓度为9%的SDS-PAGE结合分子标记(Bx7、Bx7OE和By 8)方法对陕西品种(系)检测结果表明,Glu-A1、Glu-B1和Glu-D1分别有3、8和3个亚基种类,共28种亚基组合类型。以1、Null、7+9、7+8、7+8*、14+15、2+12和5+10为主要亚基类型,频率分别为55.6%、41.6%、43.9%、26.2%、10.3%、15.4%、79.0%和12.6%;同时在Glu-B1位点发现7+8*和6+8*亚基。该方法可以快速准确地评价小麦HMW-GS组成,有效区分Glu-B1位点的亚基7与7OE,8与8*,鉴定结果稳定可靠。     

Polymerization of glutenin during grain development in near-isogenic wheat lines differing at Glu-D1 and Glu-B1 in greenhouse and field

The polymerization of glutenin polymers was monitored by measuring the Unextractable Polymeric Protein (UPP) at 3-day intervals after anthesis for four pairs of near-isogenic wheat lines. Two pairs, the variety Lance, differing at the Glu-D1 locus (HMW-GS 5+10 or 2+12) and the variety Halberd, differing at the Glu-B1 locus (HMW-GS 7+9 or 20x+20y) were grown in the field (2000) and twice in the greenhouse (2000 and 2001). Two other pairs, the varieties Warigal and Avocet, differing at the Glu-D1 locus (HMW-GS 5+10 or 2+12) were grown in the greenhouse in 2001. The behavior of all lines was consistent from greenhouse and field plantings in that the lines possessing strength-associated HMW-GS (5+10 at Glu-D1 and 7+9 at Glu-B1) showed an increase in accumulation of larger glutenin polymers (measured by UPP) at an earlier stage during grain filling than the lines with allelic counterparts (HMW-GS 2+12 at Glu-D1 and 20x+20y at Glu-B1). In all cases, the increases were maintained until maturity, paralleling the greater dough strength of flour from these lines, measured by mixograph dough development time.     

Stacking HMW-GS transgenes in bread wheat: Combining subunit 1Dy10 gives improved mixing properties and dough functionality

We have determined the technological properties of four lines containing combinations of three HMW-GS transgenes, encoding HMW-GS 1Ax1, 1Dx5 and 1Dy10. These lines were produced by conventional crossing of three single transgenic lines of the bread wheat cultivar Anza that contains the endogenous HMW-GS pairs 1Dx2 + 1Dy12 and 1Bx7* + 1By8 and is null for the Glu-A1 locus. Consequently, the total number of HMW-GS ranged from 4 in the control line Anza to 7 in line T618 which contains all three HMW-GS transgenes. The lines were studied over two years using a range of widely used grain and dough testing methods. All lines with transgenic subunits showed higher levels of glutenin proteins than the Anza control, and these differences were highly significant for lines T616, T617 and T618, containing, respectively, the transgenes encoding HMW-GS 1Ax1 and 1Dy10, 1Dx5 and 1Dy10 and 1Ax1, 1Dx5 and 1Dy10. These increases in glutenin levels are compensated by lower levels of gliadins present in transgenic lines. These changes affected the ratio of polymeric to monomeric gluten proteins (poly:mono), the ratio of HMW-GS to LMW-GS (HMW:LMW) and the contents of individual 1Ax, 1Bx, 1By, 1Dx and 1Dy subunits. Transgenic lines expressing subunit 1Dy10 together with x-type subunits (T616, T617 and T618) were superior to line T606, which had only increases in x-type subunits. In particular, the combination of transgenic subunits 1Dx5 and 1Dy10 (line T617) gave better dough rheological properties than the other combinations of transgenic subunits. For example, dough development time and stability were increased by 3.5-fold and 8.5-fold, respectively, while the mixing tolerance index (MTI) was decreased by 3.3-fold in line T617 with respect to the control line. Alveograph analyses showed that all four transgenic combinations had increased P values compared to the Anza control but subunit 1Dx5 greatly reduced the extensibility (L). These results show that stacking HMW-GS transgenes by conventional crossing is a valid strategy for the improvement of wheat quality, with different effects being related to the different HMW-GS combinations.