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Highlight Quantitative trait loci for wheat root hair length co-locate with those previously described for harvest index and thousand grain weight., Root hairs are fast growing, ephemeral tubular extensions of the root epidermis. They arise in the unsuberized maturation zone of the root, effectively increasing the root surface area in the region over which nutrient and water uptake occur. Variation in root hair length (RHL) between varieties has been shown to be genetically determined, and could, therefore, have consequences for nutrient capture and yield potential in crops. We describe the development of a medium-to-high throughput screening method for assessing RHL in wheat at the seedling stage. This method was used to screen a number of wheat mapping population parental lines for variation in RHL. Parents of two populations derived from inter-varietal crosses differed for RHL: Spark vs Rialto and Charger vs Badger. We identified quantitative trait loci (QTLs) for RHL in the populations derived from these crosses. In Spark × Rialto, QTLs on chromosomes 1A, 2A and 6A were associated with variation in RHL, whilst in Charger × Badger, a QTL for RHL was identified on 2BL. The QTLs on 2A and 6A co-localized with previously described QTLs for yield components. Longer root hairs may confer an advantage by exploiting limiting mineral and water resources. This first QTL analysis of root hair length in wheat identifies loci that could usefully be further investigated for their role in tolerance to limiting conditions.

Summary Crop yields must increase to address food insecurity. Grain weight, determined by grain length and width, is an important yield component, but our understanding of the underlying genes and mechanisms is limited. We used genetic mapping and near isogenic lines (NILs) to identify, validate and fine-map a major quantitative trait locus (QTL) on wheat chromosome 5A associated with grain weight. Detailed phenotypic characterisation of developing and mature grains from the NILs was performed. We identified a stable and robust QTL associated with a 6.9% increase in grain weight. The positive interval leads to 4.0% longer grains, with differences first visible 12 d after fertilization. This grain length effect was fine-mapped to a 4.3 cM interval. The locus also has a pleiotropic effect on grain width (1.5%) during late grain development that determines the relative magnitude of the grain weight increase. Positive NILs have increased maternal pericarp cell length, an effect which is independent of absolute grain length. These results provide direct genetic evidence that pericarp cell length affects final grain size and weight in polyploid wheat. We propose that combining genes that control distinct biological mechanisms, such as cell expansion and proliferation, will enhance crop yields.

The occurrence of late maturity alpha-amylase (LMA) was investigated using two doubled haploid wheat populations segregating for the dwarfing gene Rht-D1b and the 1B/1R translocation. Genotypes were assessed in the field and in controlled environments where a cold-shock treatment was used to induce LMA. Results from field-grown genotypes from the cross Spark × Rialto suggest that the absence of Rht-D1b or the presence of the 1B/1R translocation increases the expression of LMA.These two genetic factors were found to act independently and to have a positive interaction (complementary epistasis). In Option × Potent genotypes fixed for Rht-D1b, the 1B/1R effect was similar to that seen in the equivalent Spark × Rialto genotypes. Under controlled environment conditions, genotypes with the 1B/1R translocation showed a higher occurrence of LMA under both control and cold-shock conditions. 1B/1R was present in the majority of genotypes expressing LMA under control and cold-shock conditions. The results point to the novel finding that the 1B/1R translocation increases the expression of alpha-amylase in LMA-prone germplasm independently of effects of Rht-D1b, whereas previously it had been thought to act by a modification of the Rht-D1b effect.

Analysis of the extent of genetic variation within genetic resources is important for diversity preservation and also for breeders who exploit it. We investigated the recently introduced molecular marker technique of DNA diversity array technology (DArT), with the objective of characterising diversity in the likely relatively narrow genetic background of Czech malting barley cultivars. A total of 94 obsolete or registered barley cultivars and some hulless barley lines primarily of Czech origin were characterised by DArT analysis. A total of 271 polymorphic marker alleles were revealed across the analysed set of accessions, 37 of which were identified as being overrepresented; the other 234 markers were used for further analysis. The average dissimilarity value within the analysed set of accessions was 0.692. To assess how well DArT is suited for individual barley characteristic evaluation, available agronomical data from three yield field trials were used. Out of 94 barley genotypes used in the field trials that were assessed by DArTs, 41 have been grown over time as malting cultivars in the region. Similarity matrices based on Gower’s coefficient for mixed data and simple matching coefficient were used to compare DaRT and agronomical results. We demonstrate that a DArT-based similarity matrix and an agronomical data-based similarity matrix correlated well. To assess the genetic structure of the entire collection, K-means and simple matching coefficient clustering were used. Statistical analysis confirmed the power of the DArT system, in fact they efficiently grouped old genetic resources and modern cultivars in the expected way. Our results show that the level of genetic diversity has not changed substantially over time, but significant shifts in allelic frequency have occurred. In addition, a DArT-based dendrogram and principal component analysis (PCA) plots clearly demonstrated the impact of breeding practices on the diversity of Czech spring malting barley cultivars over time.

The location of new genes for resistance to common bunt in wheat is valuable for gene pyramiding in breeding. For this purpose, the genetics of the relatively high level of resistance in the European winter wheat variety Trintella was investigated using a doubled haploid mapping population of a cross between Trintella and the susceptible variety Piko. The population was scored for bunt infection in the field for 2 years following inoculation with a mixture of teliospores of Tilletia tritici and T. laevis. A genetic map consisting of 29 linkage groups was constructed using polymorphic simple sequence repeat markers. This map was used for QTL analysis, and in both years, results indicated that resistance to common bunt could mostly be attributed to a gene on chromosome 1B, near to the centromere and closest to marker Xgwm273 on the short arm. Additionally, in 2008, smaller QTL effects were ascribed to chromosomes 7A and 7B, and another smaller QTL effect to chromosome 5B in 2009 only.

In bread wheat (Triticum aestivum L.), crop height is an important determinant of agronomic performance. The aim of this study was to identify genes controlling variation in crop height segregating in elite European winter wheat germplasm. Four doubled haploid populations derived from the crosses Avalon × Cadenza, Savannah × Rialto, Spark × Rialto and Charger × Badger were selected, representing wide diversity in European winter wheat breeding programmes. Genetic maps based on simple sequence repeat (SSR) and Diversity Arrays Technology (DArT) markers were constructed for each population. One hundred and four significant quantitative trait loci (QTL) were identified in the four populations. A meta-analysis was conducted and the effects condensed into sixteen meta-QTL on chromosomes 1A, 1B, 1D, 2A (two meta-QTL), 2B, 2D, 3A, 3B, 4B, 4D, 5A, 5B, 6A, 6B and 6D. These include QTL with additive effects equivalent to Rht-D1 and a potentially new allele of Rht8. The description of these effects offers new opportunities for the manipulation of crop height, biomass and yield in wheat breeding programmes.

Grain morphology in wheat (Triticum aestivum) has been selected and manipulated even in very early agrarian societies and remains a major breeding target. We undertook a large-scale quantitative analysis to determine the genetic basis of the phenotypic diversity in wheat grain morphology. A high-throughput method was used to capture grain size and shape variation in multiple mapping populations, elite varieties, and a broad collection of ancestral wheat species. This analysis reveals that grain size and shape are largely independent traits in both primitive wheat and in modern varieties. This phenotypic structure was retained across the mapping populations studied, suggesting that these traits are under the control of a limited number of discrete genetic components. We identified the underlying genes as quantitative trait loci that are distinct for grain size and shape and are largely shared between the different mapping populations. Moreover, our results show a significant reduction of phenotypic variation in grain shape in the modern germplasm pool compared with the ancestral wheat species, probably as a result of a relatively recent bottleneck. Therefore, this study provides the genetic underpinnings of an emerging phenotypic model where wheat domestication has transformed a long thin primitive grain to a wider and shorter modern grain.

It is desirable to produce transgenic plants which have optimized and stable levels of transgene expression. Low levels of transgene expression may lead to an insufficient quantity of transgenic protein being produced for a particular purpose. This report demonstrates a means of enhancing transgene expression in barley beyond that conferred by the Ubi1 promoter, via the inclusion of an intron at a specific position within the transgene coding sequence. We independently cloned two different introns (RpoT-i4 from maize and UBQ10-i1 from Arabidopsis) into the same position within the firefly luciferase (luc) coding sequence. The constructs produced were transformed into barley (Hordeum vulgare) via Agrobacterium-mediated transformation, and the resulting transformant populations (of between 119 and 123 independent plants for each construct) were assayed for luciferase activity. Both introns significantly increased luciferase activity, and a quantitative reverse-transcription polymerase chain reaction assay revealed that the introns increased the accumulation of luciferase mRNA transcripts. The enhanced transgene expression levels were maintained in the T(1) and T(2) progenies. These findings show that intron-mediated enhancement is a valuable additional tool for achieving high and stable levels of transgene expression in crop plants.

Based on studies of the distribution of alleles at the important Rht and Ppd loci on wheat chromosomes 4B, 4D and 2D, different groups of winter wheat cultivars registered in the Czech and Slovak Republics during the period 1976–2007 were examined for a range of agronomic traits using official data from multi-location trials. Significant variation for all traits was detected among and between genotype groups. The frequent introduction of ‘Rht-D1b’ cultivars from the UK and Western Europe to the Czech Republic since 1995 has positively influenced lodging resistance and undoubtedly also yielding ability, but negatively affected winter-hardiness and bread making quality. An improved opportunity for earlier flowering cultivars with high winter-hardiness levels, in combination with high bread-making quality, can be obtained with genotypes carrying the Xgwm261 allele 192-bp that is probably indicative of the presence of Rht8. While GA insensitive Rht genes caused approximately a 10 cm reduction of plant height, the 192-bp allele at Xgwm261 was not associated, in these conditions, with a significant reduction in plant height when compared to Xgwm261 alleles 165- and 174-bp. Likewise, the photoperiod insensitive allele Ppd-D1a did not have a significant effect on plant height and it had not adversely affected other characters. Later heading genotypes carrying Xgwm261 alleles174- and 165-bp, often in combination with Ppd-D1b, could probably guarantee broader adaptability, which is highly desirable for changeable weather conditions. While the presence of the 192-bp allele was clearly associated with suitability for cultivation in the warmer maize growing regions, this was not so obvious for Ppd-D1a, particularly when combined with the 174-bp allele. GA responsive genes did not, apparently, influence adaptability to the different growing conditions. These studies reveal that there were both shortcomings and benefits attributable to the use of germplasm from different origins when introducing Rht and Ppd alleles. These results should be helpful to breeders in optimizing the choice of parents for crossing, and selection strategy in these target environments.

Near isogenic lines (NILs) varying for reduced height (Rht) and photoperiod insensitivity (Ppd-D1) alleles in a cv. Mercia background (rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht8c+Ppd-D1a, Rht-D1c, Rht12) were compared for interception of photosynthetically active radiation (PAR), radiation use efficiency (RUE), above-ground biomass (AGB), harvest index (HI), height, weed prevalence, lodging and grain yield, at one field site but within contrasting (‘organic’ vs. ‘conventional’) rotational and agronomic contexts, in each of 3 years. In the final year, further NILs (rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht-B1b+Rht-D1b, Rht-D1b+Rht-B1c) in Maris Huntsman and Maris Widgeon backgrounds were added together with 64 lines of a doubled haploid (DH) population [Savannah (Rht-D1b) × Renesansa (Rht-8c+Ppd-D1a)]. There were highly significant genotype × system interactions for grain yield, mostly because differences were greater in the conventional system than in the organic system. Quadratic fits of NIL grain yield against height were appropriate for both systems when all NILs and years were included. Extreme dwarfing was associated with reduced PAR, RUE, AGB, HI, and increased weed prevalence. Intermediate dwarfing was often associated with improved HI in the conventional system, but not in the organic system. Heights in excess of the optimum for yield were associated particularly with reduced HI and, in the conventional system, lodging. There was no statistical evidence that optimum height for grain yield varied with system although fits peaked at 85 and 96 cm in the conventional and organic systems, respectively. Amongst the DH lines, the marker for Ppd-D1a was associated with earlier flowering, and just in the conventional system also with reduced PAR, AGB and grain yield. The marker for Rht-D1b was associated with reduced height, and again just in the conventional system, with increased HI and grain yield. The marker for Rht8c reduced height, and in the conventional system only, increased HI. When using the System × DH line means as observations grain yield was associated with height and early vegetative growth in the organic system, but not in the conventional system. In the conventional system, PAR interception after anthesis correlated with yield. Savannah was the highest yielding line in the conventional system, producing significantly more grain than several lines that out yielded it in the organic system.

Genetic studies were conducted on an European winter wheat cultivar, Beaver, to determine the mode of inheritance of leaf rust resistance at seedling and adult plant growth stages using a recombinant doubled haploid population, Beaver/Soissons. Greenhouse studies indicated the involvement of genes Lr13 and Lr26 in governing leaf rust resistance at seedling growth stages, whereas, adult plant resistance (APR) in the field with pathotypes carrying virulence individually for Lr13 and Lr26 showed trigenic inheritance for the population. Marker regression analysis of adult plant field data indicated the involvement of six significant QTLs (chromosomes 1B, 3B, 3D, 4B, 4D and 5A) in year 2005, four QTLs (1B, 3B, 4B and 5A) in 2006, and six QTLs (1A, 1B, 3B, 4A, 4B and 5A) in 2007 for reducing leaf rust severity. QTLs on chromosomes 1B, 4B and 5A were considered the most important because of their detection across years, whereas QTLs on chromosomes 1A, 3B, 3D and 4A were either inconsistent or non-significant and unexplained. Based on an association of closely linked markers with phenotypic data, putative single gene stocks were identified for each consistent QTL and crossing was initiated to develop populations segregating for each to permit fine mapping of the identified regions.

The single chromosome substitution lines of chromosome 3B of the Czech alternative wheat variety Ceska Přesivka (CP 3B) into two spring varieties Zlatka and Sandra, revealed clear differences in flowering time compared to the recipient varieties. To map this gene(s), recombinant substitution lines for chromosome 3B were produced from crosses of the substitution lines with their recipient parents and genetic maps developed using SSR markers. Two populations were mapped, Sandra//Sandra 3B/Sandra (CP 3B) and Zlatka//Zlatka/Zlatka (CP 3B). Combining the genotype data with phenotype data on flowering time in five independent experiments under natural long day or controlled short day conditions revealed a single flowering time QTL. This gene had an additive effect of 1–6 days, depending on environment and genetic background, and was mapped in both populations to a position in the region of marker Xbarc164 near the centromere on the long arm of 3B. Comparisons of the genetic maps with other 3B maps developed by the authors indicated that the QTL may be homologous to a QTL segregating in UK germplasm.

Several sets of wheat inter-varietal chromosome substitution lines (SLs) have been produced over the last fifty years at the CRI (formerly RICP) in Prague-Ruzyně, based on cytogenetic manipulations using aneu - ploids. Lines with defined genes have been obtained which significantly influence growth habit and flowering time and these have been used particularly in the study of the genetics and physiology of flowering. The sets of lines include substitutions of homoeologous group 5 chromosomes carrying vrn genes that control vernalisa- tion response, homoeologous group 2 chromosomes with Ppd genes controlling photoperiodic sensitivity, and some other substitutions, particularly those with chromosome 3B of the Czech alternative variety Ceska Přesivka where a novel flowering time effect was located. Although the phenotypic and cytological analysis of substitution lines has been continually carried out during backcrossing generations, only the use of molecular markers can allow an unambiguous characterization to verify that substitutions are correct and complete. This analysis has allowed incorrect substitutions or partial substitutions to be identified and discarded. This paper summarizes the results of recent molecular checks of the substitution line collections at CRI.

The development of novel transformation vectors is essential to the improvement of plant transformation technologies. Here, we report the construction and testing of a new multifunctional dual binary vector system, pCLEAN, for Agrobacterium-mediated plant transformation. The pCLEAN vectors are based on the widely used pGreen/pSoup system and the pCLEAN-G/pCLEAN-S plasmids are fully compatible with the existing pGreen/pSoup vectors. A single Agrobacterium can harbor (1) pCLEAN-G and pSoup, (2) pGreen and pCLEAN-S, or (3) pCLEAN-G and pCLEAN-S vector combination. pCLEAN vectors have been designed to enable the delivery of multiple transgenes from distinct T-DNAs and/or vector backbone sequences while minimizing the insertion of superfluous DNA sequences into the plant nuclear genome as well as facilitating the production of marker-free plants. pCLEAN vectors contain a minimal T-DNA (102 nucleotides) consisting of direct border repeats surrounding a 52-nucleotide-long multiple cloning site, an optimized left-border sequence, a double left-border sequence, restriction sites outside the borders, and two independent T-DNAs. In addition, selectable and/or reporter genes have been inserted into the vector backbone sequence to allow either the counter-screening of backbone transfer or its exploitation for the production of marker-free plants. The efficiency of the different pCLEAN vectors has been assessed using transient and stable transformation assays in Nicotiana benthamiana and/or Oryza sativa.

The introgression of desirable genes or alleles from the wild relatives of hexaploid wheat can be a valuable source of genetic variation for wheat breeders to enhance modern varieties. The UK Group 1 bread making variety Shamrock is an example where the introgression of genetic material from wild emmer (Triticum dicoccoides) has been used to develop a modern cultivar. A striking character of Shamrock is its unique viridescent colour compared to other UK wheats, a trait that coincides with a non-glaucous phenotype. A doubled haploid population segregating for the trait (Shamrock × Shango) was examined to map the location of Vir, and analyse any associated pleiotropic effects. The viridescence gene located to the distal end of the short arm of chromosome 2B. QTL analysis of productivity traits shows an association between Vir and a significant delay in senescence, resulting in an extension of the grain filling period. A stable yield QTL, accounting for up to a quarter of the variation in one case, was also identified at or near Vir, indicating significant yield benefits either by linkage or pleiotropy.

Crops frequently display genotype x environment interaction for yield and end-use quality in response to different environments, particularly when stresses such as water limitation and temperature are components of the interaction. Plant breeders usually exploit this variation via phenotypic selection to develop varieties with both general and specific adaptation. However the individual genes and physiological processes underlying the basis of general and specific adaptation have rarely been elucidated. We are combining large-scale QTL analysis of several doubled haploid populations of wheat, grown over different environments and seasons, with detailed physiological analysis, to dissect the genes and mechanisms responsible for yield and yield x environment variation in adapted European winter germplasm. Analysis of populations grown under irrigated and non-irrigated conditions on drought-prone soils has revealed individual genes showing stable and differential expression over environments, and the analysis has also identified physiological traits that contribute to differential yield performance. Genes on the homoeologous group 2 chromosomes were associated with flag leaf senescence (stay-green) variation and were the most significant in drought interactions. Variation for stem soluble carbohydrate reserves was associated with the 1RS arm of the 1BL/1RS translocated chromosome, and was positively correlated with yield under both irrigated and non-irrigated conditions, and thus general adaptability. Separate analyses of populations grown over three seasons in England, Scotland, France and Germany revealed QTL for yield performance showing both general and specific effects. A stable QTL on chromosome 6A, consistent in different populations, showed significant effects over seasons and environments, whilst other QTL were specific to season and/or environments.

Greenbug and Russian wheat aphid (RWA) are two devastating pests of wheat. The first has a long history of new biotype emergence and, recently, RWA resistance has just started to break down. Thus, it is necessary to find new sources of resistance that will broaden the genetic base against these pests in wheat. Seventy-five doubled haploid recombinant (DHR) lines for chromosome 6A from the F 1 of the cross between 'Chinese Spring' and the 'Chinese Spring (Synthetic 6A) (Triticum dicoccoides x Aegilops tauschii)' substitution line were used as a mapping population for testing resistance to greenbug biotype C and to a new strain of RWA that appeared in Argentina in 2003. A quantitative trait locus (QTL) for antixenosis to greenbug was significantly associated with the marker loci Xgwm1009 and Xgwm1185 located in the centromere region of chromosome 6A. Another QTL which accounted for most of the antixenosis against RWA was associated with the marker loci Xgwm1293 and Xgwm1150, both located on the long arm of chromosome 6A. This is the first report of greenbug and RWA resistance genes located on chromosome 6A. It is also the first report of antixenosis against the new strain of RWA. As most of the RWA resistance genes present in released cultivars have been located in the D-genome, it is highly desirable to find new sources in other genomes to combine the existing resistance genes with new sources.

Lodging is a major constraint to increasing yield in many crops, but is of particular importance in the small-grained cereals. This study investigated the genetic control of lodging and component traits in wheat through the detection of underlying quantitative trait loci (QTL). The analysis was based on the identification of genomic regions which affect various traits related to lodging resistance in a population of 96 doubled haploid lines of the cross 'Milan' x 'Catbird', mapped using 126 microsatellite markers. Although major genes related to plant height (Rht genes) were responsible for increasing lodging resistance in this cross, several other traits independent of plant height were shown to be important such as root and shoot traits, and various components of plant yield. Yield components such as grain number and weight were shown to be an indicator of plant susceptibility to lodging. QTL for lodging and associated traits were found on chromosomes 1B, 1D, 2B, 2D, 4B, 4D, 6D and 7D. QTL for yield and associated traits were identified on chromosomes 1B, 1D, 2A, 2B, 2D, 4D and 6A.

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T1 populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.

Breeding for genetic resistance against greenbug and Russian wheat aphid (RWA) is the most effective way of controlling these widespread pests in wheat. Earlier work had shown that chromosome 7D of a synthetic hexaploid wheat, ‘Synthetic’ (T. dicoccoides × Ae. squarrosa) (AABB × DD) gave resistance when transferred into the genetic background of an aphid-susceptible cultivar, ‘Chinese Spring’, as the recipient. To map the genes involved, a set of 103 doubled haploid recombinant substitution lines was obtained from crossing the 7D substitution line with the recipient, and used to determine the number and chromosomal location of quantitative trait loci (QTL) controlling antixenosis and antibiosis types of resistance. Antixenosis to RWA was significantly associated with marker loci Xpsr687 on 7DS, and Xgwm437 on 7DL. Antibiosis to greenbug was associated with marker loci Xpsr490, Rc3 (on 7DS), Xgwm44, Xgwm111, Xgwm437, Xgwm121 and D67 (on 7DL). Similarly, antibiosis to RWA was linked to loci Xpsr490, Rc3, Xgwm44, Xgwm437 and Xgwm121. At least two QTL in repulsion phase, one close to the centromere either on the 7DS or 7DL arms, and a second distal on 7DL could explain antibiosis to RWA and, partially, this mechanism against greenbug.

Variation in transgene expression levels can result from uncontrolled differences in experimental protocols. It is important to quantify and eliminate this unwanted variation as much as possible in order to attain precision in transgenic studies. Large-scale transgenic studies could, by their design, generate additional variation. The influence of different plant growth, sampling and analysis strategies in generating spurious variation in transgene expression level quantification in rice plant populations was assessed. The use of multiple independent plant phenotypic analyses (enzymatic assays in this study) was identified as the major source of spurious variation (doubling or tripling the variation). The quantification of transgene expression levels was also found to be significantly influenced by plant age, the choice of leaf sampled and leaf size. All of these factors reduced the precision of molecular genetic studies and generated artefactual results in transgenic studies. Identification of the sources of extraneous variation allowed the development of a new standard operating procedure (SOP) for rice, designed to control spurious variation. SOP allowed the influence of differences in growth period and independent phenotypic analyses to be minimized. The coefficient of variation in transgene expression levels, between and within genetically identical rice plants, was reduced to approximately 10 to 15% using SOP. Adoption of quality assurance (QA) criteria such as SOP is key to improving the reproducibility of transgenic studies.

Using a pair of near-isogenic lines(NILs) of winter wheat (Triticumaestivum L.) contrasting for the Ppd-D1 and ppd-D1 alleles, in eachof Mercia and Cappelle-Desprez, experimentsin two seasons (1997/8 and 1998/9) on aloamy medium sand examined differences inflowering date, resource capture, biomassproduction and grain yield responses toirrigation. Drought did not occur for anysustained period in unirrigated conditionsin 1998 due to high seasonal rainfall. In1999, drought developed post-floweringunder unirrigated conditions. Ppd-D1on average advanced flowering by 12 days inMercia and 9 days in Cappelle-Desprez.Earlier flowering with Ppd-D1 was dueto a shorter thermal duration from cropemergence to GS31, with no effect on thethermal duration from GS31 to GS61. In bothgenetic backgrounds, Ppd-D1 decreasedabove-ground dry matter (AGDM) at harvestin irrigated conditions by 0.3–0.9 tha-1 (p< 0.05), but thiswas compensated for by increases inharvest index (HI), so that grain yield wasconserved. Although Ppd-D1 decreasedmaximum green area index (GAI) by 0.8–1.9this was countered by greater maintenanceof green area after flowering, so thatradiation interception during grain fillingwas conserved. The Ppd-D1 alleledecreased season-long crop water uptake inthe Mercia NILs in irrigated conditions by39 mm. Effects of drought in 1999,averaging across NILs, were todecrease machine-harvested grain yield by 0.6 t ha-1 in Mercia and by 1.8 tha-1 in Cappelle-Desprez (p

The timing of flag leaf senescence (FLS) is an important determinant of yield under stress and optimal environments. A doubled haploid population derived from crossing the photo period-sensitive variety Beaver,with the photo period-insensitive variety Soissons, varied significantly for this trait, measured as the percent green flag leaf area remaining at 14 days and 35 days after anthesis. This trait also showed a significantly positive correlation with yield under variable environmental regimes. QTL analysis based on a genetic map derived from 48 doubled haploid lines using amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers, revealed the genetic control of this trait. The coincidence of QTL for senescence on chromosomes 2B and 2D under drought-stressed and optimal environments, respectively, indicate a complex genetic mechanism of this trait involving the re-mobilisation of resources from the source to the sink during senescence.

The aim of this investigation was to test the developmental patterns of deletion lines, generated for chromosome arms 5AL and 5DL in the variety ‘Chinese Spring’ (CS) under vernalized and non-vernalized treatments. Plants were grown in controlled conditions under saturating daylength. Time to heading and the duration of particular phases before flowering were recorded, and leaf and spikelet production rates and numbers were analysed. The lines lacking Vrn-A1 and Vrn-D1 were delayed in time to heading under non-vernalized conditions, because of the lengthening of the emergence to floral initiation phase (EM-FI) and the terminal spikelet to heading phase (TS-H). Differences in final leaf numbers corresponded to longer durations of the EM-FI phase. The absence of Vrn-A1 and Vrn-D1 apparently decreased the number of spikelets by a lower primordium production rate, even though the duration of the FI-TS phase was longer or equal to CS. The sensitivity to vernalization in lines where the Vrn genes were deleted was much higher.

Transgenic lines of the spring barley variety Golden Promise containing the firefly luciferase gene were produced by particle bombardment of immature embryos. Non-destructive analysis of luciferase gene expression was used to monitor the transformation process. This revealed that transformation efficiency, in terms of the percentage of bombarded immature embryos giving rise to transformed callus lines, was very high, up to 40%. Following the expression of the luciferase gene provided a method for the sensitive, non-destructive, real-time monitoring of gene expression throughout the transformation process. Luciferase expression could also be used to easily identify transgenic plants and to identify homozygous transgenic plants at an early stage. The production of transgenic barley by selecting for luciferase-positive material, without an additional selection system, was possible but technically difficult.

The temporal and spatial expression of three promoters was investigated in transgenic rice plants using promoter-β-glucuronidase ( gusA ) reporter gene fusions. The promoters studied were ubiquitin-1 (UBI-1) of Zea mays , Cauliflower Mosaic Virus 35S gene (CaMV35S) and a tubulin gene (TUB-1) of Arabidopsis thaliana . The TUB-1 promoter provided 7.32-fold more GUS activity in roots relative to tillers. This was significantly different from the corresponding value of 2.82-fold for CaMV35S but not from that of 4.55-fold for UBI-1, activity of both promoters was higher in the root tips and zone of elongation than mature roots. This younger root tissue represented a declining proportion of the expanding root system with time. Older tissue expressing GUS under control of the TUB-1 promoter showed a steeper decline in activity with time than occurred with the UBI-1 promoter. Nematode infection did not alter the overall pattern of expression from the two promoters, except that the giant cells induced by Meloidogyne incognita retained TUB-1 promoter activity as roots matured. Pratylenchus zeae invaded older root regions than M. incognita and no changes in promoter activity were detected where it fed. The results suggest the TUB-1 promoter has characteristics that favour its use for delivering anti-feedants, such as cysteine proteinase inhibitors, to M. incognita .

The relationship between grain protein concentration and grain yield in different cultivars of winter wheat was examined in a series of field experiments carried out over three years, in which 13, 12 and 8 cultivars were studied in each year, respectively. The plants were grown at sites located in Shropshire, west-central England, in years 1 and 2, and at three other locations in eastern England in year 3. Above ground plant samples were collected at an thesis and again at maturity, when they were separated into grain and straw, and analysed for dry matter and N content. Analysis of residuals from regression of grain protein concentration on grain yield (grain protein deviation, GPD) showed that some cultivars had a higher grain protein concentration than was predicted from grain yield alone. It was deduced that the capacity to accumulate a higher grain protein concentration than predicted from grain yield is under genetic control and thus may be improved through breeding. Other factors (weight of N accumulated in the biomass at anthesis, weight of N accumulated in the biomass between anthesis and maturity and the concentration of N remaining in the straw at maturity) were added step-wise into the regression to enable statistical analysis of their relative contributions to grain protein. High GPD may be achieved through increased N accumulation after anthesis, combined with efficient re-translocation of vegetative N reserves. The use of GPD provides a selection criteria in wheat breeding programs to screen for increased grain protein concentration without a concurrent grain yield reduction.

Using some of the chromosome substitution lines developed from the crosses of the donor Cheyenne to Chinese Spring we showed that the accumulation of water soluble carbohydrates during different stages of hardening was time dependent. More over there was a significant correlation between the rate of carbohydrate accumulation and the frost tolerance. The expression and regulation of a wheat gene homologous to the barley cold regulated corl4b gene was compared in frost sensitive and frost tolerant wheat genotypes at different temperatures. Studies made with chromosome substitution lines showed that the threshold induction temperature polymorphism of the cor14b wheat homologous gene was controlled by loci located on chromosome 5A of wheat, while corl4b gene was mapped, in Triticum monococcum, onto the long arm of chromosome 2A“’. Our study on the effect of cold hardening on glutathione (GSH) metabolism showed that chromosome 5A of wheat has an influence on the GSH accumulation and on the ratio of reduced and oxidised glutathione as part of a complex regulatory function during cold hardening. In addition, the level of increase in GSH content during hardening may indicate the degree of the frost tolerance of wheat.

The development of comprehensive genetic maps based on molecular markers has increased the power of genetical analysis immensely in the last few years. Characters previously recalcitrant to analysis, such as abiotic stress responses, are now amenable, and individual major genes and QTL mediating the variation can be identified. This has allowed the development of strategies for stress amelioration by adjusting the timing of the life cycle and introducing genes which enable the plant to tolerate stress. These strategies are illustrated here by showing how genes for vernalization response and cold tolerance on chromosomes 5A and 5D of wheat have been identified and located. Additionally, their relationships to genes in other species, such as barley and rice can be characterised through comparative mapping approaches, leading to strategies for their isolation using rice genomic tools.

Barbela is an old Portuguese landrace of wheat that is highly genetically heterogeneous. Different Barbela populations when subjected to aluminium stress show variable levels of tolerance. In order to study the inheritance of this character, doubled haploid (DH) lines were developed. These DH were obtained by intergeneric crosses of 14 different lines of Barbela with maize. During this process the efficiency of the technique was evaluated and suggestions for its improvement were obtained. Several parameters were studied in the crosses: % of crossability, % of embryos per florets pollinated and % of embryos per seed set. The different genotypes of Barbela showed significant variation for the parameters analysed. When the reciprocal crosses were analysed, no differences were found, indicating that cytoplasm differences do not influence the parameters of DH production. However, different spikelet positions (lower, middle and upper) gave highly significant differences in all parameters analysed. Highest success frequencies were obtained for pollinated spikelets in the middle of the spike. This can indicate that concentrating on the middle part of the spike can increase the frequency of DHs obtained using inter generic crosses of wheat with maize.

Immature embryos of the spring barley variety GoldenPromise, were bombarded with three different particledelivery systems and both transient and stabletransformation examined. In addition, a range oftechniques for the preparation of the DNA coated goldparticles was examined. Fertile transgenic barleyplants were obtained using three particle preparationtechniques which differed in the amount of gold andDNA used for each bombardment. However, only one ofthe particle delivery systems, the PDS 1000/He device,appeared to be effective in yielding transformedbarley plants.

A restriction fragment length polymorphism (RFLP) based linkage map of a cross between two diploid Hordeum bulbosum (2n = 2x = 14) clones, PB1 and PB11, was constructed from 46 recombinant progeny clones. Since both parents are heterozygous, separate and combined parental maps were constructed. All of the RFLP markers screened had previously been mapped in barley (H. vulgare L.) so that comparative maps could be produced. The PB1 linkage map consists of 20 RFLP marker loci assigned to four linkage groups covering 94.3 cM. The PB11 linkage map consists of 27 RFLP marker loci assigned to six linkage groups covering 149.1 cM. Thirteen markers polymorphic in both parents were used as ‘anchors’ to create a combined linkage map consisting of 38 loci assigned to six linkage groups and covering a genetic distance of 198 cM. Marker order was highly conserved in a comparison with the linkage map of H. vulgare (Laurie etal., 1995). However, in contrast, the genetic distances for the same markers were very different being 649 cM and 198 cM respectively, a genetic distance ratio of 1: 3.3. Thus although the map was short, it can be presumed to cover half the genome of H. bulbosum. This study provides further confirmation of the close relationship between the two species and gives a basis for the development of marker mediated introgression through interspecific hybridisation between the two species.

Transformed rice plants of var `TN1' were regenerated from immature embryos following particle bombardment with a construct containing the firefly luciferase gene as a reporter gene and the hygromycin resistance gene as a selectable marker. Expression of the luciferase gene in the presence of the substrate luciferin was visualised in the calli derived from bombarded immature embryos and in the leaves and roots of the regenerated transformed plants using a low light imaging system (luminograph). Embryogenic callus proliferation and plant regeneration were unaffected by luciferin treatment and luminograph screening. The quantitative Luc assay using samples of leaf tissue from the segregating generations gave early information about the homozygous and hemizygous state of the luc transgene.

The present work was initiated to assess tissue culture techniques as a means of generating and selecting herbicide tolerant genotypes of wheat through exploiting somaclonal variation. Callus was initiated from immature embryos of genetically defined varieties and subcultured onto selective media containing 5 μm, 10 μm and 50 μm concentrations of difenzoquat and atrazine. Plants were regenerated from all the selective media except that media containing the highest herbicide concentration. The progenies of the regenerated plants were tested as whole plants for their response to spray application of the herbicides. For difenzoquat, variation in response from extreme susceptibility to tolerance was observed. However, genetic characterization by progeny testing tolerant lines revealed that the induced variation was not heritable. No plants tolerant to atrazine were obtained. Overall, no clear evidence of heritable mutations was obtained. Alternative strategies to obtain herbicide tolerant genotypes using cell culture techniques are discussed.

Grain yield (GY) and grain protein concentration (GPC) are two major traits contributing to the economic value of the wheat crop. These are, consequently, major targets in wheat breeding programs, but their simultaneous improvement is hampered by the negative correlation between GPC and GY. Identifying the genetic determinants of GPC and GY through quantitative trait loci (QTL) analysis would be one way to identify chromosomal regions, allowing improvement of GPC without reducing GY using marker-assisted selection. Therefore, QTL detection was carried out for GY and GPC using three inter-connected doubled haploid populations grown in a large multi-environment trial network. Chromosomes 2A, 2D, 3B, 7B and 7D showed co-location of QTL for GPC and GY with antagonistic effects, thus contributing to the negative GPC-GY relationship. Nonetheless, genomic regions determining GPC independently of GY across experiments were found on chromosomes 3A and 5D and could help breeders to move the GPC-GY relationship in a desirable direction.

The advent of molecular marker systems has made it possible to develop comparative genetic maps of the genomes of related species in the Triticeae. These maps are being applied to locate and evaluate allelic and homoeoallelic variation for major genes and quantitative trait loci within wheat, and to establish the pleiotropic effects of genes. Additionally, the known locations of genes in related species can direct searches for homoeologous variation in wheat and thus facilitate the identification of new genes. Examples of such analyses include the validation of the effects of Vrn1 on chromosome 5A on flowering time in different crosses within wheat; the indication of pleiotropic effects for stress responses by the Fr1 locus on chromosome 5A; the detection of homoeologous variation for protein content on the homoeologous Group 5 chromosomes; and the detection of a new photoperiod response gene Ppd-H1 in barley from homoeology with Ppd2 of wheat.

Several statistical methods which employ multiple marker data are currently available for the analysis of quantitative trait loci (QTL) in experimental populations. Although comparable estimates of QTL location and effects have been obtained by these methods, using simulated and real data sets, their accuracy and reliability have not been extensively investigated. The present study specifically examines the merit of using F2 and doubled haploid populations for locating QTL and estimating their effects. Factors which may affect accuracy and reliability of QTL mapping, such as the number and position of the markers available, the accuracy of the marker locations and the size of the experimental population used, are considered. These aspects are evaluated for QTL of differing heritabilities and locations along the chromosome.

Heptaploid hybrids between octoploid triticale and wheat were backcrossed as female parents with wheat to examine the rye chromosome distribution in the resultant progenies using genomic in situ hybridization (GISH). One hundred and one backcross (BC) seeds were examined and whole rye chromosome additions and substitutions, wheat/rye centric and noncentric translocations and rye telocentric chromosomes were detected. Dicentric wheat/rye translocated chromosomes were also observed. Comparisons were made with previous results on the rye chromosome distribution from male gametes of the same cross and differences were found, where in the female derived population a deficit of plants with more than two rye chromosomes was apparent relative to the anther derived population.

The rate of organ emergence and the duration of developmental phases are key components of plant fitness to its environment. Here, we describe a hybrid genetic algorithm for the estimation of the parameters of complex non-linear simulation models that we used to estimate the varietal parameters of a well-evaluated ecophysiological model (Sirius) of wheat phenology. The aim of this study was to analyze the correlations between the varietal parameters of Sirius and to determine the minimum number of parameters that need to be estimated in order to accurately simulate the effect of the genotype and environment on wheat anthesis date. A panel of 16 bread wheat cultivars was grown under controlled conditions with different vernalization and daylength treatments and in a field nursery with different sowing dates in order to characterize their vernalization and photoperiodic requirements and earliness per se. These cultivars were also grown in the field in two consecutive growing seasons in France and in the UK along a 800 km latitude transect, where the dynamics of leaf appearance and anthesis date were determined. Variation in both final leaf number and anthesis date in response to the environment and the genotype was predicted with a mean error of 0.55 leaves and 3.94 d, respectively, after estimation of only three of the seven varietal parameters of Sirius: the phyllochron, the response of vernalization rate to temperature, and the daylength response of leaf production. Among them, the phyllochron was reasonably well estimated and showed a positive association with earliness per se (Spearman's coefficient of rank correlation = 0.59). This study showed that the number of varietal parameters in Sirius was overestimated and that considering only three varietal parameters reduced the correlations between the parameters and the root mean square error of prediction for final leaf number and anthesis date. We conclude that a phenomenogical model of wheat development can be used to estimate key phenological parameters that are difficult to determine in the field; offering the possibility to conduct large-scale quantitative genetic studies to understand better the genetic control of flowering time in cereals.

Summary A number of stably transformed wheat cell lines were obtained using two different direct gene transfer techniques. When integration of the foreign genes was investigated in DNA samples taken at 10 months post-selection, complicated profiles of transgene bands were observed in Southern blot analysis. Among these, a number of common bands were identified showing similar hybridization patterns between independently transformed cell lines. This type of hybridization pattern has been a common observation and is usually interpreted as concatameric rearrangement of integrated DNA. However, when integration was reinvestigated with DNA samples taken at 30 months postselection, the hybridization pattern changed and most of the common bands had disappeared. Further analysis using a set of methylation-sensitive enzymes revealed that the DNA represented by the common bands was N6-adenine methylated (m6A DNA), and there was even m6A DNA in some 30 month samples. Although the source of m6A DNA in the wheat cultures was not clearly established, the data indicate that transformation of an endophyte (e.g. a mycoplasma-like organism) may have occurred at the same time as the transformation of wheat cells. The integration pattern of undermethylated transgenes in the transformed cells became simpler and clearer after treating the DNA preparations with Dpnl, which only cuts m6A DNA. The implications of these data for other methods of inoculating cereals with DNA are discussed.

RFLP analysis of 105 doubled haploid lines from a cross between the barley varieties ‘Magnum’ and ‘Goldmarker’ located the denso dwarfing gene on the long arm of chromosome 3(3H), approximately 8 cM distal to the RFLP locus Xpsrl70. Lines with the denso gene showed a distinctive prostrate juvenile growth habit and tended to have later ear emergence times and lower plant grain weights, ear grain weights and 50 grain weights.

The composition of the high-molecular-weight (HMW) subunits of glutenin encoded by Glu-1 in hexaploid wheat (Triticum aestivum L.) was determined for 99 varieties bred in China. Seventy-nine varieties were homogeneous for HMW glutenin subunit composition. The majority of the remainder were heterogeneous at one Glu-1 locus, with two varieties heterogeneous at two loci. One variety carried a pattern of subunits not encountered in previous studies. The majority of varieties, as in previous studies, carried subunits 7+8 or 7+9 encoded by Glu-B1. In contrast, there was a relatively high occurrence of subunits 2+12 at Glu-D1. It was shown that a high proportion, 69%, of the potential variation in bread-making quality, as assessed by SDS-sedimentation volume, was accounted for by variation in the composition of the HMW subunits.

A novel methodology is described in which transcriptomics is combined with the measurement of bread-making quality and other agronomic traits for wheat genotypes grown in different environments (wet and cool or hot and dry conditions) to identify transcripts associated with these traits. Seven doubled haploid lines from the Spark x Rialto mapping population were selected to be matched for development and known alleles affecting quality. These were grown in polytunnels with different environments applied 14 days post-anthesis, and the whole experiment was repeated over 2 years. Transcriptomics using the wheat Affymetrix chip was carried out on whole caryopsis samples at two stages during grain filling. Transcript abundance was correlated with the traits for approximately 400 transcripts. About 30 of these were selected as being of most interest, and markers were derived from them and mapped using the population. Expression was identified as being under cis control for 11 of these and under trans control for 18. These transcripts are candidates for involvement in the biological processes which underlie genotypic variation in these traits.

Recent advances in crop research have the potential to accelerate genetic gains in wheat, especially if co-ordinated with a breeding perspective. For example, improving photosynthesis by exploiting natural variation in Rubisco's catalytic rate or adopting C(4) metabolism could raise the baseline for yield potential by 50% or more. However, spike fertility must also be improved to permit full utilization of photosynthetic capacity throughout the crop life cycle and this has several components. While larger radiation use efficiency will increase the total assimilates available for spike growth, thereby increasing the potential for grain number, an optimized phenological pattern will permit the maximum partitioning of the available assimilates to the spikes. Evidence for underutilized photosynthetic capacity during grain filling in elite material suggests unnecessary floret abortion. Therefore, a better understanding of its physiological and genetic basis, including possible signalling in response to photoperiod or growth-limiting resources, may permit floret abortion to be minimized for a more optimal source:sink balance. However, trade-offs in terms of the partitioning of assimilates to competing sinks during spike growth, to improve root anchorage and stem strength, may be necessary to prevent yield losses as a result of lodging. Breeding technologies that can be used to complement conventional approaches include wide crossing with members of the Triticeae tribe to broaden the wheat genepool, and physiological and molecular breeding strategically to combine complementary traits and to identify elite progeny more efficiently.

Near isogenic lines (NILs) varying for genes for reduced height (Rht) and photoperiod insensitivity (Ppd-D1a) in a cv. Mercia background (rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht8c + Ppd-D1a, Rht-D1c, Rht12) were compared at one field site but within contrasting (‘organic’ vs. ‘conventional’) rotational and agronomic contexts, in each of 3 years. In the final year, further NILs (rht (tall), Rht-B1b, Rht-D1b, Rht-B1c, Rht-B1b + Rht-D1b, Rht-D1b + Rht-B1c) in both Maris Huntsman and Maris Widgeon backgrounds were added together with 64 lines of a doubled haploid (DH) population [Savannah (Rht-D1b) × Renesansa (Rht-8c + Ppd-D1a)]. Assessments included laboratory tests of germination and coleoptile length, and various field measurements of crop growth between emergence and pre jointing [plant population, tillering, leaf length, ground cover (GC), interception of photosynthetically active radiation (PAR), crop dry matter (DM) and nitrogen accumulation (N), far red: red reflectance ratio (FR:R), crop height, and weed dry matter]. All of the dwarfing alleles except Rht12 in the Mercia background and Rht8c in the DHs were associated with reduced coleoptile length. Most of the dwarfing alleles (depending on background) reduced seed viability. Severe dwarfing alleles (Rht-B1c, Rht-D1c and Rht12) were routinely associated with fewer plant numbers and reduced early crop growth (GC, PAR, DM, N, FR:R), and in 1 year, increased weed DM. In the Mercia background and the DHs the semi-dwarfing allele Rht-D1b was also sometimes associated with reductions in early crop growth; no such negative effects were associated with the marker for Rht8c. When significant interactions between cropping system and genotype did occur it was because differences between lines were more exaggerated in the organic system than in the conventional system. Ppd-D1a was associated positively with plant numbers surviving the winter and early crop growth (GC, FR:R, DM, N, PAR, height), and was the most significant locus in a QTL analysis. We conclude that, within these environmental and system contexts, genes moderating development are likely to be more important in influencing early resource capture than using Rht8c as an alternative semi-dwarfing gene to Rht-D1b.

BackgroundPlant transformation is an invaluable tool for basic plant research, as well as a useful technique for the direct improvement of commercial crops. Barley (Hordeum vulgare) is the fourth most abundant cereal crop in the world. It also provides a useful model for the study of wheat, which has a larger and more complex genome. Most existing barley transformation methodologies are either complex or have low (ResultsA robust, simple and reproducible barley transformation protocol has been developed that yields average transformation efficiencies of 25%. This protocol is based on the infection of immature barley embryos withAgrobacteriumstrain AGL1, carrying vectors from the pBract series that contain thehptgene (conferring hygromycin resistance) as a selectable marker. Results of large scale experiments utilising theluc(firefly luciferase) gene as a reporter are described. The method presented here has been used to produce hundreds of independent, transgenic plant lines and we show that a large proportion of these lines contain single copies of thelucgene.ConclusionThis protocol demonstrates significant improvements in both efficiency and ease of use over existing barley transformation methods. This opens up opportunities for the development of functional genomics resources in barley.

Biotic and abiotic stresses are responsible for much of the variation in wheat production worldwide. The plant, in responding to stress events, increases the endogenous synthesis of hormones such as Abscisic Acid, Ethylene, Jasmonic Acid and Salicylic Acid. This paper was aimed at determining the location of plant-defence genes triggered by treatment with stress-induced hormones in a set of recombinant doubled haploid substitution lines involving variation for a single chromosome, 6A, previously shown to carry genes for aphid resistance. Eighty-three doubled haploid recombinant substitution (DHR) lines for chromosome 6A derived from the F1 of “Chinese Spring” (CS) × “Chinese Spring (Synthetic 6A)” (S6A) substitution line, were used as a mapping population. Plants of every genotype at the fully expanded 3rd leaf stage were subjected to four hormone treatments: ethylene (E), jasmonic acid (J), salicylic acid (S) and abscisic acid (A), together with an untreated control. The changes in a range of phenotypic traits were measured, aerial fresh weight (AFWC), aerial dry weight (ADWC), root fresh weight (RFWC), root dry weight (RDWC), foliar area (FAC). These were recorded twice: at the onset of the experiment before spraying with hormones and 72 h later. Nine QTLs were detected which were significantly associated with 6A molecular markers, explaining the variation for ADWC, RDWC and FAC. Most of the QTLs were associated with the interval between loci Xgwm459 and Xgwm334a, located in the telomeric region of the short arm of 6A. The dissection of complex agronomic traits such as tolerance to stress and QTLs related to exogenous hormone treatments could be used in marker-assisted selection for breeding wheat with tolerance to stresses.

Durum wheats cultivated in the Middle Anatolia region of Turkey (Triticum durum cvs 'Kunduru','Berkmen' and 'Cakmak') were crossed with maize to evaluate their capacity for haploid embryo and doubled haploid (DH) plant production. A total of 2960 florets were crossed with maize and 13.7% of the florets produced haploid embryos across the three varieties. Haploid embryo and plant regeneration frequencies were highest in 'Kunduru', but colchicine doubling was less successful with this genotype. There were statistically significant differences between 'Cakmak' and other genotypes for embryo yield (P

Variation in transgene expression levels can result from uncontrolled differences in experimental protocols. Studies conducted over generations could, by their design, generate additional unwanted variation. To study sources of spurious variation, transgene expression levels were quantified over five homozygous generations in two independent transgenic rice lines created by particle bombardment. Both lines contained the same gus expression unit and had been shown to exhibit stable inheritance of transgene structure and expression. All plants were cultured and sampled using previously developed standardized protocols. Plants representative of each generation (T2, T3, T4, T5, T6) were grown either all together or across several different growth periods. GUS activity in plants from different generations was quantified either in the same assay or over multiple independent assays. Strategies in which plants were grown and phenotyped independently, significantly increased (up to 3-fold) extraneous variation in transgene expression level quantification, thus reducing the precision of molecular genetic studies and generating artefactual results in transgenic studies conducted over generations. Identification of sources of unwanted variation and quantification of their effect allowed the development of new strategies designed to control spurious variation. Growth and phenotyping of all plants from all generations together, using standard operating procedures (SOP), led to a reduction in extraneous variation associated with transgene expression level quantification. Adoption of such strategies is key to improving the reproducibility of transgenic studies conducted over generations.