Session 4 Biochemistry, Cytogenetics, Transgenic Gene

Oral Presentation

Genetic Transformation of Barley: Improved Technology,
Safety Assessment and Future Potential

W. A. Harwood1, L. J. Bilham1, S. Travella1,2, V. Bourdon1,3, H. Salvo-Garrido1,4, J. Harden1, M. Perry1 and J. W. Snape1

1John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK,
E-mail: wendy.harwood@bbsrc.ac.uk; 2Institute of Plant Biology, University of Zurich, 8008 Zurich, Switzerland; 3Department of Plant Sciences, University of Oxford, Oxford, OX1 3RB, UK;
4Biotechnology Unit (INIA), Carillanca, Temuco, Chile

Reliable methodologies are now available for the genetic transformation of barley using either direct DNA delivery by particle bombardment, or more recently, by Agrobacterium-mediated gene delivery. We are now focusing on the Agrobacterium-mediated system whose advantages include higher efficiency, lower transgene copy number, more predictable expression and the possibility of producing clean-gene transgenic lines where the selectable marker can be segregated away from the gene of interest. Even with improved transformation methods, transgene expression is still unpredictable. Large variations in transgene expression levels may be seen between different transgenics with the same construct, and between generations of the same transgenic line. Some variation in expression may be attributed to so called position effects due to the genomic environment of the transgene. By combining analysis of the physical position of transgenes (using fluorescent in situ hybridisation) with knowledge of their genetic map positions, and detailed molecular analysis including determining transgene flanking regions, we can increase our understanding of factors affecting transgene expression levels in barley. This analysis indicates that active transgenes may not be inserted at random in the host genome. Assessment of the performance of GM barley lines, compared to appropriate controls, under field conditions is essential in addition to extensive analysis of glasshouse grown material. We have examined GM barley lines over five generations under field conditions where we have measured variation in target and non-target gene expression.

Dissecting Large Genomes of Triticeae by Chromosome Sorting

J. Dolezel, M. Kubalakova, P. Suchankova, J. Safar, J. Janda, P. Kovarova, J. Bartos,
J. Cihalikova and H. Simkova

Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany, 772 00 Olomouc, Czech Republic; E-mail: dolezal@ueb.cas.cz

Nuclear genomes of Triticeae are characterized by large size and prevalence of repetitive DNA sequences. These features hamper physical mapping and gene isolation. Purification of individual chromosomes by flow cytometry greatly simplifies these tasks by providing small and defined genome fractions. This lecture will review the development of the methodology and its potential for genome mapping in barley, rye and wheat. Due to small differences in relative DNA content, only one chromosome type may be discriminated and sorted in each of the three species. Cytogenetic stocks facilitate separation of other parts of the genomes as individual chromosomes, translocated chromosomes and chromosome arms. Chromosome analysis by flow cytometry permits quantitative detection of structural and numerical chromosome changes. Sorted chromosome fractions have been used for discovery of rare structural changes and high-resolution cytogenetic mapping using FISH. The use of sorted chromosomes for HAPPY mapping, targeted isolation of genic sequences, and higher-throughput physical mapping of ESTs on microarrays are attractive options. As millions of chromosomes with intact DNA may be sorted, construction of BAC libraries is possible. Subgenomic, chromosome-specific and chromosome arm-specific BAC libraries have already been constructed in wheat and represent unique resources for wheat genomics.

Supported by the Ministry of Education, Youth and Sports of the Czech Republic (Grants No. ME527 and ME528).

Molecular Analysis and its Utilization in Barley Breeding

W. Powell, K. MacKenzie, J. R. Russell, K. Caldwell, A. Booth, I. Hein, P. E. Hedley
and W. T. B.Thomas

Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
E-mail: wpowel@scri.sari.ac.uk

The primary goal of plant breeding programmes is to identify recombination events that confer a beneficial phenotypic effect. Mendelian genetics provides the foundation for distinguishing between genotype and phenotype together with the hereditary principles that allow statistical testing of independent assortment and linkage. Meiosis and recombination based analysis was first discovered in plants and the main practical beneficiary of these discoveries was plant breeding. My presentation will focus on how genetic analysis will facilitate barley breeding and the need to ensure that the catalogues of genes emerging from gene-discovery programmes can provide functional alleles for deployment in barley breeding. I will illustrate these trends with reference to input (disease resistance) and output (quality) traits and emphasise the critical need to obtain robust phenotype data and the value of population development for genomics based approaches to gene isolation and validation. Understanding the patterns of intra-specific sequence diversity will be an important tool to create haplotype based approaches for gene localisation and validation of function. This information when coupled with a knowledge of barley breeding populations will allow: improved annotation of germplasm, a better understanding of germplasm dynamics and the ability to monitor and capture significant recombination events in the form of elite germplasm. Genetic analysis of complex traits will require integrated approaches based on both meiotic and physical mapping. Exploitation of new knowledge emerging from these areas of endeavour will require considerable ingenuity in which the role of the barley breeder will be paramount.

SSR Markers in Barley Breeding and Genomics

P. Langridge1, M. Pallotta1 and M. Hayden2

1Australian Centre for Plant Functional Genomics and 2Molecular Plant Breeding CRC, University
of Adelaide, Glen Osmond, SA 5064, Australia, E-mail: peter.langridge@adelaide.edu.au

The use of molecular markers to track loci and genome regions in barley is routinely applied in many breeding programs. The location of major loci is now known for many disease resistance genes, tolerances to abiotic stresses and quality traits. There are three aspects that are particularly significant. First, we now have markers closely linked to many traits of importance in the breeding programs. Indeed we have markers for more loci than we can screen in a conventional breeding program. Second, we have tools that allow marker scanning of the whole genome. SSR markers form the base for this analysis and highly multiplexed SSR screens have been developed for barley. This has improved our capacity for whole genome screens. Third, through association mapping projects we have or are in the process of developing, whole genome fingerprints for many key lines and varieties of importance in breeding. Existing strategies for marker-assisted selection (MAS) were initiated with a view of markers as providing a rapid and cheap alternative to bioassays and they have largely been used in this role. While highly successful, this strategy does not fully utilize the technology. The key challenge of new work is to investigate strategies for whole genome breeding: that is, to see how we can use genome information to design optimal breeding strategies that integrate as much of the available information as possible.

The Barley Microarray A Community Vision
and Application to Abiotic Stress

T. j. Close

Department of Botany and Plant Sciences, University of California, 92521 USA,
E-mail: timothy.close@ucr.edu

A barley microarray chip representing approximately 22,000 barley genes has been produced as part of a USDA-IFAFS project entitled, An integrated physical and expression map of barley for Triticeae improvement. The content of the chip was derived from more than 400,000 barley EST sequences received from cooperators in USA, Germany, Japan, Scotland, and Finland, plus about 1,000 sequences retrieved from the GenBank nr database. All EST sequences were trimmed to high quality regions, contaminants were identified and removed, and the remaining information was compiled using the CAP3 assembly program. A stringent assembly (paralogs separated) contained a total of about 53,000 unigenes (the sum of contigs plus singletons), among which about 50% had reliable 3 ends. From these, probe sets were designed and the chip fabricated by Affymetrix. Details on the content of the barley microarray can be viewed using the free software HarvEST:Barley, which is available from http://harvest.ucr.edu. The availability of an Affymetrix barley microarray has facilitated the study of gene expression on a large scale. Biologically triplicated experiments on drought, salinity, low temperature and ABA-treatment have revealed commonalities and differences between responses to abiotic stresses, and inherent differences between genotypes

Transgenic Barley

D. von Wettstein

Department of Crop and Soil Sciences, School of Molecular Biosciences and Center of Integrated Biotechnology, Washington State University, Pullman, WA 99164-6420, USA,
E-mail: diter@wsu.edu

Genetic transformation of scutellum cells of immature zygotic barley embryos is routine using either particle bombardment or co-cultivation with Agrobacterium tumefaciens. In a few cultivars regeneration of the scutellum cells via callus and somatic embryos into plants has yielded frequencies of more than 50% transformants. For expression of desired genes during grain germination/malting alpha-amylase promoters are preferred together with the code of the transit peptide for secretion of the expressed protein into the endosperm. For deposition of microbial and human enzymes and growth factors in the storage protein bodies of the mature grain, the hordein gene promoters can deliver up to 1 g of recombinant protein per kg of grain. The herbicide resistant gene required for selection of a transformant can be eliminated with double cassette vectors or by site directed recombination. The genetically stable transformants have generally decreased grain production and thousand-grain weights. As with spontaneous and induced mutations, decreased agronomic performance is rectified by marker assisted recombination breeding with elite cultivars. The transgene insertion site in the barley genome at the nucleotide sequence level is determined with a modified tail PCR procedure. Maize Ac/Ds transposons have been inserted into the barley genome for gene tagging. Greenhouse and field trials with genetically engineered stem rust and Rhizoctonia root rot resistant transformants will be reviewed. Addition of 0.02% grain containing a protein engineered, thermotolerant (1,3-1,4)--glucanase to normal barley is sufficient to achieve in feeding trials equal weight gain of broilers as obtained with corn diets.

Poster Presentation

Functional Analysis of Hordatines and Identification
of Mutants in their Biosynthesis in Barley

A. K. Batchu, P. Schulze-Lefert and T. Koprek

Department of Plant-Microbe Interactions, Max-Planck Institute of Plant Breeding, D-50829 Cologne, Germany, E-mail: batchu@mpiz-koeln.mpg.de

Plants often contain anti-microbial compounds which are either performed or synthesized upon pathogen infection. Expanding knowledge of the biological function of these metabolites, their mode of action and the regulation of their biosynthesis are essential for the understanding of plant-microbe interactions on the molecular level. One of these secondary compounds, the hordatines, are dimers of hydroxy cinnamoyl agmatines. Though the cinnamic acid amine conjugates are common in cereals and some other plant species, hordatines are exclusively found in some representatives of the genus Hordeum. We have investigated the temporal and spatial distribution and the biological activity of these metabolites. We found that hordatines are abundant in the young seedling and possess strong antifungal activity in vitro. Information about the in planta function and the regulation of the biosynthetic pathway is limited. Preliminary data suggests that hordatines serve as preformed anti-fungal compounds in H. vulgare as they could act in cellwall strengthening to restrict the pathogen infection and they may function as cytotoxic components during hypersensitive response. In order to shed light on the function of hordatines in planta, it is necessary to identify mutants in hordatine biosynthesis. We devised a high-throughput screening method based on MALDI-TOF mass spectrometry which allows a rapid screening of mutant populations with high accuracy.

Haplotype Diversity and Recombination in the asi Gene of Barley

P. C. Bundock and R. J. Henry

Molecular Plant Breeding Cooperative Research Centre, Centre for Plant Conservation Genetics,
Southern Cross University, Lismore NSW 2480, Australia,
E-mail: pbundock@scu.edu.au

The asi gene (alpha amylase/subtilisin inhibitor) of barley is involved in seed defence against pathogens. It is a single copy intronless gene that is expressed in maternal tissues of the seed. We have obtained sequence from sixteen barley genotypes of 2,164 bp of DNA including promoter, transcribed and downstream regions of the asi gene. The genotypes sequenced consisted of four wild barley accessions from Israel, a Saharan landrace and eleven modern cultivars. Eighty SNPs and nineteen indels were detected in addition to repeat length polymorphism at a microsatellite site in the promoter region. Six major haplotypes could be distinguished, with two haplotypes representing the diversity among the eleven cultivated barleys and the other four haplotypes belonging to the wild and landrace accessions. A minimum of seven recombination events explain the related patterns of variation between the haplotypes. Effective recombinations in a largely selfing species would be much rarer than in outcrossing species and comparisons with results from studies of other genes in wild barley indicate that the asi gene may be a hotspot for recombination.

Gene Expression Characterization of the Barley Morphological Mutant Leafy Lemma through cDNA Microarray Analysis
of Isogenic Lines Derived from lel × Kaskade Cross

P. Faccioli, F. Rizza, A. M. Stanca and V. Terzi

Experimental Institute for Cereal Research, 29017 Fiorenzuola dArda (PC), Italy,
E-mail: p.faccioli@iol.it

Leafy lemma barley mutant carries recessive alleles of two genes, both of which are necessary to cause the transformation of the lemma into a structure having all characteristics of a vegetative leaf, as shown by SEM analysis. Mapping positions of the two lel genes on barley chromosomes 5 and 7 has been determined using F2 populations derived from leafy lemma × Nudinka crosses. Starting from the cross leafy lemma × Kaskade, isogenic lines were developed: each pair of lines shows the wild type and mutant version of lemma in the same genetic background. The characterization of the isogenic lines was done evaluating a set of agronomic traits in two years trials and measuring photosynthetic efficiency of the leafy lemma structure in comparison with other photosynthetic tissues of the plant. Gene expression was studied and compared in leafy lemma, awn and flag leaf from pairs of isogenic lines using microarray technology. EST clones from a barley leaf cDNA library were arrayed and used to test differences in mRNA expression level in the mutant lemma in comparison with other photosynthetic tissues of the plant. To support the microarray-based results, the variation in hybridization signals in some clones was confirmed by real time RT-PCR analysis.

Production of Recombinant Gelatin in Barley

H. Holkeri1, E. Wahlstroem2, A. Ritala1, J. Baez3, K. Maekinen2 and A. M. Nuutila1

1VTT Biotechnology, Espoo, FIN-02044, Finland,
E-mail: anneli.ritala@vtt.fi;
2Department of Applied Biology, University of Helsinki, FIN-00014 Helsinki, Finland; 3FibroGen Inc.,
South San Francisco, CA 94080, USA

Collagen and gelatin are attractive mammalian protein models for expression in plants because their use in the health care industry requires a safer, more homogeneous product than the currently animal-derived material. Additionally, collagen requires a complex post-translational modification and triple helical assembly. The aim of the project is to develop a production system for properly processed recombinant collagen and gelatin using barley seed as a production host. Two strong transcriptional promoters will be used for production of recombinant collagen in transgenic barley during germination and maturation of barley seeds. The 5-end of the Cocksfoot mottle virus (CfMV; genus Sobemovirus) genomic RNA sequence, called CfMV -element, has been shown to enhance transient recombinant protein synthesis in barley cells. We will study whether it further increases accumulation levels of collagen in transgenic plants. A mammalian prolyl-4-hydroxylase will be co-expressed with collagen. Appropriate hydroxylation of collagen is required for the thermal stability of the triple helical conformation of collagen at body temperature.

Analysis of Patterns of Gene Expression during Barley Grain Development Using Serial Analysis of Gene Expression (SAGE)

A. F. M. Ibrahim, P. E. Hedley, L. Cardle, D. F. Marshall and R. Waugh

Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK,
E-mail: rwaugh@scri.sari.ac.uk

Serial Analysis of Gene Expression (SAGE) is a very powerful technique which can provide quantitative data concerning gene expression without the requirement for pre-existing sequence information. It provides both the qualitative information for the identification of differentially expressed genes in a particular cell population and quantitative information for the identification of differentially expressed genes in different cell populations or under different developmental or environmental conditions. We are applying the SAGE technology to the analysis of gene expression during barley grain development. The data presented here represent results obtained from three SAGE libraries. The first library was constructed from whole developing grain at 12 days post anthesis (dpa) and sequences of 51,963 SAGE tags were obtained, representing ca. 14,500 expressed genes. Detailed analysis of the 100 most highly represented tags is described. A second library was generated from a different RNA preparation from the same tissue and used to statistically verify the reproducibility of the obtained results. A third library was generated from dissected embryos at a similar stage (12 dpa), providing the basis for a more detailed analysis of gene expression within one stage of grain development. The data presented here provide the first results towards a genome-wide view of gene expression during barley grain development.

Proteomic Analyses of Mature Barley Seeds among Recombinant Chromosome Substitution Lines Generated from a Cross between Hordeum vulgare subsp. spontaneum and H. v. subsp. vulgare
cv. Harrington

Q. Jiang, D. Roche, S. Sorensen, J. Waverly and D. Hole

Plants, Soils and Biometeorology Department, Utah State University, Logan, UT 84322-4820, USA,
E-mail: droche@mendel.usu.edu

Using two-dimensional electrophoresis we are in the process to characterized seed proteins of recombinant chromosome substitution lines (RCSLs) derived from a cross and backcrosses between Hordeum vulgare subsp. vulgare (cv. Harrington as recurrent parent) and H. v. subsp. spontaneum (as donor parent) (Matus et al., Genome, 46, 2003: 10101023). This RCLS mapping population gives us the opportunity to monitor the effects of chromosome substitution on the recurrent proteome of mature Harrington seeds. We found a significant number of polymorphic hydrophilic proteins between seed extracts of the recurrent and donor parental lines. From whole seed extracts of Harrington cv. and H. v. spontaneum, we resolved 287 hydrophilic protein spots in a narrow pH gradient (5.56.7). Only 92 of them are present in both lines indicating a large qualitative level of polymorphism. Among these 92 matched spots, normalized spot intensities for 17 and 18 of them further indicate a differential representation of three-fold decrease and three-fold increase, respectively. The quantitative data from Harrington whole seeds is used as reference gel (internal control) for the analyses of extracts from RCSLs. We will discuss prospects to expedite the comparison of barley seed proteomes with fluorescence (2D-DIGE) tools or Isotope Coded Affinity Tags (ICAT TM).

Differential Expression Proteomics in Barley and its Wild Relatives

K. Kakeda1, Y. Hanai1 and K. Sato 2

1Faculty of Bioresources, Mie University, Tsu 514-8507, Japan; 2Barley Germplasm Center,
Research Institute of Bioresources, Okayama University, Kurashiki 710-0046, Japan,
E-mail: kazsato@rib.okayama-u.ac.jp

Large amounts of EST (Expressed Sequence Tag) sequences have been available in barley. A cDNA microarray technique using ESTs is a powerful tool for the large-scale comparative analysis of gene expression. However, gene expression profiles at the mRNA level are often different from those at the protein level. Post-transcriptional modifications and degradations of proteins also influence the expression of genes. These indicate the importance of proteome approaches to study differential expression of genes. Two-dimensional (2D) gel electrophoresis and mass spectrometry (MS) techniques are most commonly used for differential expression proteomic analysis as mentioned above. Recently an alternative technique, isotope-coded affinity tagging (ICAT) has been developed. This technique, coupled with MS, enables the rapid identification and quantification of proteins expressed differently between, e.g., genotypes, tissues, developmental stages, etc. In this paper, we describe the application of the ICAT method for differential analysis of protein expression in barley and its wild relatives, and compare its feasibility with the conventional 2D gel method.

Exclusive Generation of True-Breeding Transgenic Plants via Agrobacterium-Mediated Transformation of Barley Pollen Cultures

J. Kumlehn, S. Broeders and V. Valkov

Plant Reproductive Biology, Institute of Plant Genetics and Crop Plant Research (IPK),
D-06466 Gatersleben, Germany,
E-mail: kumlehn@ipk-gatersleben.de

Methods used to identify and clone genes and promoters which are of scientific interest or have putative relevant function for plant breeding are becoming increasingly powerful. Therefore, reliable and efficient systems for genetic transformation of important plant species are needed for comprehensive functional gene analyses as well as for crop improvement approaches. Agrobacterium-mediated transformation of pollen cultures has been used for efficient barley transformation in our lab, yet only a few plants which are true-breeding with regard to the transgene can be directly generated by this method and tedious as well as time-consuming segregation analyses are necessary to phenotypically distinguish these individuals from hemizygous plants. Pursuing a novel concept, haploid primary transgenic regenerants which appear to be useless in the first instance were routinely subjected to induced genome doubling. As a result, almost all of these plants showed partial or full seed set. Although the inflorescences from many colchicine-treated plants appear to be ploidy-chimaeric, the caryopses obtained are always homozygous for the transgene, since fertile flowers can only be formed from diploid (doubled haploid) cellular origines. Thanks to stable hereditary transmission and expression of the transgene, exclusive generation of true-breeding transgenic plants will substantially contribute to increased efficiency, reproducibility and reliability of overexpression, knock-out and mutant complementation experiments.

Mainstem Leaf Development during Tillering in Spring Barley

V. Momcilovic1, N. Przulj1, D. Knezevic2 and L. Stosic3

1Institute of Field and Vegetable Crops, Novi Sad, 21000 Serbia and Montenegro,
E-mail: przulj@ifvcns.ns.ac.yu;
2ARI SERBIA Small Grains Research Centre, 34000 Kragujevac, Serbia and Montenegro;
3Postgraduate student, Faculty of Agriculture, Novi Sad, Serbia and Montenegro

Time interval between appearances of successive leaves is designated as phyllochron interval (PI). The objective of this study was to evaluate PI of mainstem leaf (MSL) development during tillering of spring barley. Six spring barley varieties were grown in Novi Sad, Serbia and Montenegro (45°20 N, 15°51 E, 86m a.s.l) from 1999 to 2001. Phyllochron was estimated using Haun scale where growing-degree-days (GDD) served as time scale. PI ranged from 60.5 GDD in the fifth to 75.8 GDD in the third leaf. All three factors, i.e., year, variety, and their interaction, were included in phyllochron determination of the first leaf. The highest percentage of variance component for first leaf phyllochron belonged to year. Variation in phyllochron of the second leaf was controlled by variety and interaction year x variety, variations in phyllochron of the third, fourth, and fifth leaf by year and interaction, and variation in the sixth leaf by interaction only. The linearity suggests that MSL stage can be used as a predictive measure of plant development and can retroactively show the quality of the preemergent seedbed environment.

Barley Population Structure and Adaptability with Small Radiation Doses Effects

N. V. Proskurnin and R. V. Kryvoruchenko

Department of Genetics, Plant Breeding and Seed-Growing, National Agrarian University
named after V. V. Dokuchaev, Kharkiv, 62483, Ukraine, E-mail: plant_biology@agrouniver.kharkov.com

In result of perennial observation of radiation hormesis effects in spring barley populations it was estimated that low doses of radiation induce a diversity of reactions on different biological organization levels (cellular, organism, population) among consistent postradiational generations. Radiation hormesis reactions induced by low doses have system character and pronounced adaptive direction. It was discovered that irradiation of barley in low doses (0.51.0 Gy) causes increase of heterogeneity of populations structural organization among generations (M1M3). Use of many-dimensional analysis methods (cluster, factor, discriminant) allowed to reveal individual plant groups in structure of experimental populations of barley, with different type of epigenetic organization of morphogenesis. Literary and own data analysis allows starting a proposal about epigenetic nature of low doses effects in barley populations. It was determined that to low doses radiation action in contrast to high doses modification of structural organization of population adaptability take place in increases direction. It supposes that low and high radiation doses produce fundamentally different genetic processes in barley populations. Possibility of line selection from experimental populations M2 with low irradiation doses which are characterize by heightened total adaptability and modified complex of quantitative trait is showed.

Cell Division Rate of Developing Barley Grain: Growing
Condition Effect

A. Rajala1, P. Peltonen-Sainio1 and M. Salmenkallio-Marttila2

1Department Crops and Biotechnology, MTT Agrifood Research Finland, Plant Production
Research Unit, FIN-31600 Jokioinen, Finland,
E-mail: ari.rajala@mtt.fi;
2VTT Biotechnology, Espoo, FIN-02044, Finland

In northern growing conditions, development and growth of spring barley is very intensive. Whether this swiftness is prominent also in grain development is not well known. To study the day length effect on post-pollination cell division. rate, five commonly cultivated barley cultivars (Barke, Kustaa, Luberon, Scarlett, SW Wikingett) were grown in Finland, Estonia and France. Repeated grain sample collection was carried out at post-pollination (1, 2, 4, 8, 16 and 21 days) to measure cell division rate. Trend of cell division rate was similar in all locations. Highest cell number was recorded at 8 days after pollination. Hence, the potential grain size, i.e. storage capacity, is determined very early at grain filling phase in all locations. Recorded maximum cell number tend to be higher in a long day conditions. Furthermore, in a short day, cell division rate was relatively slower and required more temperature accumulation to reach the same level as in a long day conditions. Prevailing growing conditions during grain filling period likely influence the packing efficiency of carbohydrates in to the endosperm cells and hence, determine the final grain size.

Identification of Differential Photosynthetic-Related Changes in Response to Temperature and Light Intensity in a Winter and a Spring Barley Genotype and in the Derived Segregating Doubled-Haploid Population

F. Rizza1, L. Guidi2, E. DeglInnocenti, L. Cattivelli1 and E. Francia1

1Experimental Institute for Cereal Research, 29017 Fiorenzuola dArda (PC), Italy,
E-mail: fulvia.rizza@libero.it;
2Dipartimento di Chimica e Biotecnologie Agrarie, Universita degli Studi di Pisa, 56124 Pisa, Italy

The winter cold-resistant parent Nure is an Italian two-rowed feeding barley cultivar, while the spring and cold-sensitive parent Tremois is a French two-rowed malting variety. The response of the two genotypes have been characterised under several conditions of acclimation and stress to abiotic factors (chilling and freezing temperature, high light intensity, high temperature) using chlorophyll fluorescence and gas exchange analysis. The genotypes were contrasting not only for different levels of stress tolerance acquired after acclimation but also for a different capacity to induce earlier responses to stress conditions; this was especially evident in variation of chlorophyll fluorescence parameters at high light intensity associated to acclimation temperature and in different sugars accumulation in leaves and roots at low temperature. A segregating population of doubled haploid (DH) lines was derived by anther culture from the F1 generation of the winter × spring barley cross Nure × Tremois, and a first analysis of a total of 136 doubled-haploid lines has been carried out.

Timing of Barley Microspore Transformation Relative to Cell Cycle

Y. S. Shim and K. J. Kasha

Department of Plant Agriculture, University of Guelph, Guelph, Ontario, N1G 2W1, Canada,
E-mail: kkasha@uoguelph.ca

Our objective is to determine the time of DNA synthesis during the uninucleate development stage of the barley microspore and, knowing that, determine if we could obtain homozygous transgenic plants by timing the introduction of foreign DNA by particle bombardment. We have been able to correlate the morphology of the uninucleate microspore with time of DNA synthesis and are able to apply an inductive pretreatment to hold the microspores at the G1 stage. We are also examining the progeny from microspores bombarded at various microspore stages and following various pretreatments to determine if plants homozygous for the insert are obtained. Plants from bombarded microspores are being selected by GFP expression in early embryogenic stages in cultures and are being examined by GFP expression, PCR analysis and FISH.

In Silico Expression Analysis of Barley ESTs: Tissue-Specificity
of Starch Accumulation and Mobilization

N. Sreenivasulu, H. Zhang, V. Radchuk, U. Wobus and W. Weschke

Department of Molecular Genetics, Institute of Plant Genetics and Crop Research (IPK),
D-06466 Gatersleben, Germany,
E-mail: srinivas@ipk-gatersleben.de

To provide resources for functional genomics studies of the development of barley caryopses, approximately 40,000 ESTs were produced from four cDNA libraries covering pre-storage (HZ, HA: maternal and filial tissues, respectively, 07 Days After Flowering, DAF), middle storage (HB: whole barley seeds, 815 DAF) and late storage phase (HF: whole barley seeds, 1625 DAF). About 36% of the ESTs have no match to sequences in Arabidopsis functional gene catalogues, suggesting that a reasonable number of transcripts are specific for monocots and/or barley. StackPack clustering of the 40,000 barley seed ESTs resulted in the identification of 4,782 tentative unigene consensi (TUCs) and 7,005 singletons, altogether a set of 11,787 unigenes. By computing abundantly expressed ESTs, correlated expression patterns of genes belonging to specific pathways were inferred for specific developmental stages during seed development. Further data mining results indicate that, while metabolism of starch synthesis and storage proteins dominates during middle storage phase (815 DAF), lipid metabolism genes are highly expressed during late storage phase (1625 DAF). Multiple alignments of TUCs allowed us to estimate the number of genes in gene families responsible for starch metabolism. To identify key regulators of starch-, storage protein- and lipid metabolism, we surveyed the abundantly expressed putative transcription factors as well as kinases during storage and maturation phase.

Advances in Flow Cytogenetics of Barley

P. Suchankova1, M. Kubalakova1, P. Kovarova1, J. Bartos1, J. Cihalikova1, T. R. Endo2
and J. Dolezel1

1Laboratory of Molecular Cytogenetics and Cytometry, Institute of Experimental Botany,
772 00 Olomouc, Czech Republic; E-mail: dolezel@ueb.cas.cz; 2Laboratory of Plant Genetics,
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan

Flow cytogenetics is an attractive tool that facilitates genome mapping. Flow cytometric analysis permits classification of isolated chromosomes according to their DNA content. As thousands of chromosomes are analysed in each sample, quantitative detection of structural and numerical chromosome changes is possible. Due to similarity in relative DNA content, only the smallest barley chromosome 1H may be discriminated and sorted. Cytogenetic stocks facilitate sorting of those translocation chromosomes, which differ in DNA content from the remaining chromosomes. Although sorted fractions of translocation chromosomes were found useful for mapping of DNA sequences to subchromosomal regions, their use remains limited. Here we report on the use of wheat-barley addition lines containing barley chromosome arms. Due to the small size, the arms may be discriminated and sorted at high purity. This advance makes barley flow cytogenetics an attractive tool that may greatly facilitate genome mapping. A possibility to purify large quantities of individual chromosome arms opens avenues for targeted isolation of genic sequences, preparation of specific probes for screening EST arrays, HAPPY mapping, and construction of chromosome arm-specific BAC libraries.

Seeds of wheat-barley addition lines were obtained from the National BioResource Project (Japan). Supported by the Ministry of Education, Youth and Sports of the Czech Republic (Grants No. ME527 and ME528).

A Rapid Method for Isolating High Quality RNA
from Freeze-Dried Tissue in Barley

P. Warner, P. Langridge and K. H. Oldach

Australian Centre for Plant Functional Genomics, University of Adelaide, Glen Osmond, SA 5064, Australia,
E-mail: klaus.oldach@adelaide.edu.au

Differential gene expression can be used for characterizing novel genes or for gaining information about biological processes on a genomic scale. To understand the function of a gene we need to know when and where a particular gene is being expressed, level of gene expression and other possible candidate genes co-regulated with the gene of interest. Microarray analysis and quantitative real-time PCR were used to analyse the expression of genes in different tissues from a number of barley and wheat genotypes under varying environmental conditions. RNA is very sensitive to degradation and therefore leaf material for RNA extraction is commonly stored at 70C or at higher temperatures if treated with commercially available RNA stabilization solutions (i.e. RNAlater, Ambion Inc., UK). In this paper, we present a simple and rapid method for extracting high quality RNA from lyophilised tissue. This protocol was established to increase sample through-put and to help alleviate storage problems. Material was sampled from different barley tissues, freeze-dried and either processed immediately or after several months of storage at room temperature and at 20C. The same extraction protocol was applied to all samples stored at the various temperatures. The quality and yield of extracted RNA was compared to RNA extracted using plant material stored at 70C by Northern blot analysis and RT-PCR.

Variability of Oil Content in By-Products of Australian Barley

J. M. Washington and A. J. Box

School of Agriculture and Wine, University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia, E-mail: jennifer.washington@adelaide.edu.au

Processing, such as pearling and malting of barley, results in significant quantities of by-product that is either sold for minimal return or is discarded at cost to the producer. Barley oil has been shown to significantly lower total serum cholesterol and LDL-C without lowering HDL-C in humans with hypercholesterolemia. High concentrations of barley oil have been found in flour pearled from barley (PF) and in brewers grain (BG), therefore extraction of oil from these by-products may be a viable, value-adding process. In this study we measured the oil content of various genotypes of barley to determine if differences could be found among barley destined for the pearling and malting markets. The total lipid (TL) content of whole barley (WB) has previously been estimated at 3% and occurs mainly in the aleurone and germ layers, mostly as free lipids in the form of triglycerides. There have been few reports of significant variation of lipid content between genotypes of barley including the cultivar Riso 1508 (4.1%TL), and some hulless waxy genotypes when compared to normal starch covered types. In this paper we discuss the lipid content of WB and PF from Australian hulless waxy genotypes and other Australian cultivars and also from BG of cv. Torrens hulless barley. Significant variation was found between the samples and higher concentrations of free lipids were found in PF of hulless waxy genotypes.

Expression of Heterologous Vitreoscilla Hemoglobin in Barley

A. Wilhelmson1, P. T. Kallio2, K.-M. Oksman-Caldentey1 and A. M. Nuutila1

1VTT Biotechnology, Espoo, FIN-02044 VTT, Finland,
E-mail: annika.wilhelmson@vtt.fi;
2Institute of Biotechnology, ETH-Zurich, 8093 Zurich, Switzerland

Anaerobiosis occurs in seeds of many higher plants during imbibition. After successful germination followed by penetration of the enclosing structures by the radicle, the embryo switches to aerobic energy metabolism. The microbial population naturally present on the covering layers of the grain may add to the oxygen deficiency by competing for oxygen. Although imbibed barley grains can survive several days of anoxia, they require oxygen to complete germination. The advances in genetic engineering have opened up new possibilities to study germination. As a metabolic engineering approach to alleviate adverse effects of inadequate oxygen availability in germinating barley, the vhb gene from the gram-negative bacterium Vitreoscilla was transferred to barley. Heterologous expression of Vitreoscilla haemoglobin (VHb) has previously been shown to improve growth properties and productivity of various microbes and plants.

Variation of Starch Pasting Properties among Lines
and Pearled Fractions of Waxy Barley Determined
by the Rapid-Visco Analyzer (RVA)

T. Yanagisawa1, E. Domon2, M. Fujita3, C. Kiribuchi-Otobe3 and T. Takayama1

1National Agricultural Research Center for Western Region (WeNARC), Zentsuji,
Kagawa, 765-8508, Japan,
E-mail: tyanagi@affrc.go.jp;
2National Agricultural Research Center for Kyusyu and Okinawa Region (KONARC),
Nishigoshi, Kikuchi, Kumamoto, 861-1192, Japan; 3National Institute of Crop Science,
Tsukuba, Ibaraki, 305-8518, Japan

Starch pasting properties and apparent amylose content of waxy barley lines (indigenous waxy lines and artificial waxy mutant) were measured by Rapid-Visco Analyzer (RVA). Apparent amylose content varied from 0 to 10%. Some indigenous waxy lines with higher amylose content showed stable hot paste viscosity. In indigenous waxy lines, RVA patterns differed between different pearled fractions perhaps because apparent amylose content in outer parts of endosperm is higher than that of inner parts. Starch pasting properties were influenced by various factors such as starch molecular size, unit chain length distribution, branching pattern, degree of phosphate substitution, granule size and distribution, lipids and protein content, and amylopectin crystallinity. To reduce the influence of those factors, near-isogenic lines for waxy gene were used to analyze pasting properties and uneven distribution of amylose in indigenous waxy barley endosperm.

French Qualification of Malting Barley

P. Boivin1, N. Ouarnier1, P. Brignon2 and J.-L. Delatte3

1Institut Français des Boissons de la Brasserie Malterie, 54512 Vandoeuvre les Nancy Cedex, France, E-mail: patrick.boivin@ifbm-qualtech.com; 2TEPRAL, 67200 Strasbourg, France; 3Malteries Soufflet,
10400 Nogent sur Seine, France

In France, the registration of a new barley variety is very structured. Three main organisations take part in the process. First, there is the CTPS (Permanent Technical Committee of Breeding) which is involved in the registration on the French catalogue and on the malting barley variety list. Then there is the CBMO (Beer Malt Barley Committee) which is involved in the testing for registration on the Malteurs and Brasseurs de France preferred variety list. Finally, IFBM is the technical tool for all tests (barley technical analyses, micromalting, pilot malting and brewing, and analyses on produced malts, worts and beers). IFBM is certified by BVQI for ISO9002 and Qualtech Laboratory is accredited for barley, malt and beer analyses by the French Accreditation Committee (COFRAC). Such a system is aimed at improving barley varieties by selecting them according to measurable data. Choice criteria depend on the Maltster and Brewer needs taking expectations of all the barley-malt-beer chain into account. The variety selection is based on barley technical analyses (moisture, proteins, extract, diastasic power and dormancy), Agronomic Index AI (yield, diseases), Quality Index QI (extract, Kolbach Index, diastasic power, wort viscosity, final attenuation), Functional Index FI (modification, friability, -glucans, -amylase, FAN), pilot productions from barley grown on the same location to beer (600 kg/20 hl). Each year an official list for malting barley is published by French Brewers & Maltsters Associations to sum up the results of the work done.

Environmental Modulation of Barley Malting Quality

J. L. Molina-Cano1, J. S. Swanston2, M. Moralejo1, J. P. Polo1, A. Rubio3 and A. W. MacGregor4

1Centre UdL-IRTA, 25198 Lleida, Spain, E-mail: joseluis.molina@irta.es; 2Scottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, UK; 3Damm Brewing Company, Malthouse, Bell-lloc, Lleida, Spain; 4Retired from Grain Research Laboratory, Winnipeg, Manitoba, Canada

The mutant TL43 and its parent genotype Triumph were grown at different seasons in Lleida (Spain) and Dundee (UK), the mutant showed higher protein and C- and D-hordein contents at all environments, but its B-hordein content showed crossover GE interaction, being higher in Scotland and lower in Spain. When studying water uptake, it was concluded that although both genotype and environment influenced it, the effect of the environment was higher, and this conclusion was linked to the hordein composition. A reappraisal of the differences between Iberian- and Nordic-grown barleys was carried out with the malting cultivar Scarlett. The most obvious difference found between them was the effect of barley total and insoluble -glucans. They were an effective barrier in the North, but appeared to increase extract in the South. A conclusion was that the positive effect of -glucans in the Iberian barleys was a consequence of their greater capacity to synthesise and release -glucan hydrolases during germination, so increasing extract yield. Another study was carried out to analyse the differences between Spanish- and Canadian-grown barleys, to which extent four cultivars were used. Canadian-grown barleys had significantly lower contents of grain protein and all-three hordein fractions than the Spanish ones, they had also higher malt yield, wort -glucan and viscosity but lower fine- and coarse-ground malt extract yield, friability, free amino nitrogen, Kolbach index, -amylase and diastatic power. In summary, the Spanish-grown barleys produced higher quantities of degrading enzymes (amylolytic, proteolytic and citolytic) during germination, thus being able to attain higher extract levels.