Functional Validation in the Triticeae

Part of the Plant Genetics and Genomics: Crops and Models book series (PGG, volume 7)


The need to discover and confirm gene function directly in both wheat and barley is growing in importance. In this chapter we outline three of the most common approaches – TILLING, ‘transient’ and ‘stable’ transformation – that are being adopted by Triticeae researchers to meet this objective. As these approaches have different outcomes, they are therefore applicable to different situations depending upon the research questions being addressed. Here, we summarise recent developments in each of these functional validation strategies, and where possible use examples to illustrate the power of each approach.


Powdery Mildew High Molecular Weight Glutenin Subunit Virus Induce Gene Silence Barley Stripe Mosaic Virus Antisense ODNs 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. Ahlquist, P., Dasgupta, R. and Kaesberg, P. (1984) Nucleotide sequence of the brome mosaic virus genome and its implications for viral replication. J. Mol. Biol. 172, 369–383.PubMedGoogle Scholar
  2. Altpeter, F., Vasil, V., Srivastava, V. and Vasil, I. K. (1996) Integration and expression of the high-molecular-weight glutenin subunit 1Ax1 gene into wheat. Nat. Biotech. 14, 1155–1159.PubMedGoogle Scholar
  3. Altpeter, F., Varshney, A., Abderhalden, O., Douchkov, D., Sautter, C., Kumlehn, J., Dudler, R. and Schweizer, P. (2005) Stable expression of a defense-related gene in wheat epidermis under transcriptional control of a novel promoter confers pathogen resistance. Plant Mol. Biol. 57, 271–283.PubMedGoogle Scholar
  4. Alvarez, M. L., Guelman, S., Halford, N. G., Lustig, S., Reggiardo, M. I., Rybabushkina, N., Shewry, P. R., Stein, J. and Vallejos, R. H. (2000) Silencing of HMW glutenins in transgenic wheat expressing extra HMW subunits. Theor. Appl. Genet. 100, 319–327.Google Scholar
  5. Ashburner, M. (1990) Drosophila: A Laboratory Handbook. Cold Spring Harbor, NY, USA.Google Scholar
  6. Ayliffe, M. A., Pallota, M., Langridge, P. and Pryor, A. J. (2007) A barley activation tagging system. Plant Mol. Biol. 64, 329–347.PubMedGoogle Scholar
  7. Azevedo, C., Sadanandom, A., Kitagawa, K., Freialdenhoven, A., Shirasu, K. and Schulze-Lefert, P. (2002) The RAR1 interactor SGT1, an essential component of R gene-triggered disease resistance. Science 295, 2073–2076.PubMedGoogle Scholar
  8. Barcelo, P., Hagel, C., Becker, D., Martin, A. and Lorz, H. (1994) Transgenic cereal (Tritordeum) plants obtained at high efficiency by microprojectile bombardment of inflorescence tissue. The Plant J. 5, 583–592.Google Scholar
  9. Barro, F., Rooke, L., Békés, F., Gras, P., Tatham, A. S., Fido, R. J., Lazzeri, P., Shewry, P. R. and Barcelo, P. (1997) Transformation of wheat with HMW subunit genes results in improved functional properties. Nat. Biotech. 15, 1295–1299.PubMedGoogle Scholar
  10. Beecher, B., Bettge, A., Smidansky, E. and Giroux, M. J. (2002) Expression of wild-type pinB sequence in transgenic wheat complements a hard phenotype. Theor. Appl. Genet. 105, 870–877.PubMedGoogle Scholar
  11. Bieri, S., Potrykus, I. and Futterer, J. (2000) Expression of active barley seed ribosome-inactivating protein in transgenic wheat. Theor. Appl. Genet. 100, 755–763.Google Scholar
  12. Bieri, S., Mauch, S., Shen, Q. H., Peart, H., Devoto, A., Casais, C., Ceron, F., Schulze, S., Steinbiß, H. H., Shirasu, K. and Schulze-Lefert, P. (2004) RAR1 positively controls steady state levels of barley MLA resistance proteins and enables sufficient MLA6 accumulation for effective resistance. The Plant Cell 16, 3480–3495.PubMedGoogle Scholar
  13. Blechl, A. E. and Anderson, O. D. (1996) Expression of a novel high-molecular-weight glutenin subunit gene in transgenic wheat. Nat. Biotech. 14, 875–897.PubMedGoogle Scholar
  14. Bliffeld, M., Mundy, J., Potrykus, I. and Futterer, J. (1999) Genetic engineering of wheat for increased resistance to powdery mildew disease. Theor. Appl. Genet. 98, 1079–1086.Google Scholar
  15. Bommineni, V. R., Jauhar, P. P. and Peterson, T. S. (1997) Transgenic durum wheat by microprojectile bombardment of isolated scutella. J. Hered. 88, 475–481.Google Scholar
  16. Brigneti, G., Martin-Herna´ndez, A. M., Jin, H., Chen, J., Baulcombe, D. C., Baker, B. and Jones, J. D. G. (2004) Virus-induced gene silencing in Solanum species. The Plant J. 39, 264–272.Google Scholar
  17. Brueggeman, R., Rostoks, N., Kudrna, D., Kilian, A., Han, F., Chen, J., Druka, A., Steffensen, B. and Kleinhofs, A. (2002) The barley stem rust resistance gene Rpg1 is a novel disease-resistance gene with homology to receptor kinases. Proc. Natl. Acad. Sci. USA 99, 9328–9333.PubMedGoogle Scholar
  18. Bruun-Rasmussen, M., Madsen, C. T., Jessing, S. and Albrechtsen, M. (2007) Stability of Barley stripe mosaic virus-induced gene silencing in barley. Mol. Plant-Microbe Interact. 20, 1323–1331.PubMedGoogle Scholar
  19. Büschges, R., Hollricher, K., Panstruga, R., Simons, G., Wolter, M., Frijters, A., van Daelen, R., van der Lee, T., Diergaarde, P., Groenendijk, J., Töpsch, S., Vos, P., Salamini, F. and Schulze-Lefert, P. (1997) The barley Mlo gene: A novel control element of plant pathogen resistance. Cell 88, 695–705.PubMedGoogle Scholar
  20. Caldwell, D. G., McCallum, N., Shaw, P., Muehlbauer, G. J., Marshall, D. F. and Waugh, R. (2004) A structured mutant population for forward and reverse genetics in barley (Hordeum vulgare L.). The Plant J. 40, 143–150.Google Scholar
  21. Castillo, A., Vasil, V. and Vasil, I. K. (1994) Rapid production of fertile transgenic plants of rye (Secale cereale L.). Bio/Technol. 12, 1366–1371.Google Scholar
  22. Cheng, M., Fry, J. E., Pang, S., Zhou, H., Hironaka, C., Duncan, D. R., Conner, T. W. and Wan, Y. (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol. 115, 971–980.PubMedGoogle Scholar
  23. Cho, M. J., Choi, H. W., Buchanan, B. B. and Lemaux, P. G. (1999) Inheritance of tissue specific expression of barley hordein promoter-uidA fusions in transgenic barley plants. Theor. Appl. Genet. 98, 1253–1262.Google Scholar
  24. Cho, M. J., Choi, H. W., Jiang, W., Ha, C. D. and Lemaux, P. G. (2002) Endosperm-specific expression of green fluorescent protein driven by the hordein promoter is stably inherited in transgenic barley (Hordeum vulgare) plants. Physiol. Plantarum 115, 144–151.Google Scholar
  25. Christensen, A. H. and Quail, P. H. (1996) Ubiquitin promoter-based vectors for high level expression of selectable and/or screenable marker genes in monocotyledonous plants. Trans. Res. 5, 213–218.Google Scholar
  26. Curtis, M. D. and Grossniklaus, U. (2003) A GATEWAY cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462–469.PubMedGoogle Scholar
  27. Dagle, J. M., Weeks, D. L. and Walder, J. A. (1991) Pathways of degradation and mechanism of action of antisense oligonucleotides in Xenopus laevis embryos. Antisense Res. Dev. 1, 11–20.PubMedGoogle Scholar
  28. Dalmais, M., Schmidt, J., Le Signor, C., Moussy, F., Burstin, J., Savois, V., Aubert, G., Brunaud, V., de Oliveira, Y., Guichard, C., Thompson, R. and Bendahmane, A. (2008) UTILLdb, a Pisum sativum in silico forward and reverse genetics tool. Genome Biol. 9, R43.PubMedGoogle Scholar
  29. De Jong, W. and Ahlquist, P. (1995) Host-specific alterations in viral RNA accumulation and infection spread in a brome mosaic virus isolate with an expanded host range. J. Virol. 69, 1485–1492.PubMedGoogle Scholar
  30. Dias, N. and Stein, C. A. (2002) Antisense Oligonucleotides: Basic Concepts and Mechanisms. Mol. Cancer Ther. 1, 347–355.PubMedGoogle Scholar
  31. Ding, X. S., Schneider, W. L., Chaluvadi, S. R., Mian, M. A. R. and Nelson, R. S. (2006) Characterization of a Brome mosaic virus strain and its use as a vector for gene silencing in monocotyledonous hosts. Mol. Plant-Microbe Interact. 19, 1229–1239.PubMedGoogle Scholar
  32. Ding, Y. and Lawrence, C. E. (2001) Statistical prediction of single-stranded regions in RNA secondary structure and application to predicting effective antisense target sites and beyond. Nucleic Acids Res. 29, 1034–1046.PubMedGoogle Scholar
  33. Douchkov, D., Nowara, D., Zierold, U. and Schweizer, P. (2005) A high-throughput gene-silencing system for the functional assessment of defense-related genes in barley epidermal cells. Mol. Plant-Microbe Interact. 18, 755–761.PubMedGoogle Scholar
  34. Edwards, M. C. (1995) Mapping of the seed transmission determinants of barley stripe mosaic virus. Mol. Plant-Microbe Interact. 8, 906–915.PubMedGoogle Scholar
  35. Fang, Y. D., Akula, C. and Altpeter, F. (2002) Agrobacterium-mediated barley (Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA:genomic DNA junctions. J. Plant Physiol. 159, 1131–1138.Google Scholar
  36. French, R., Janda, M. and Ahlquist, P. (1986) Bacterial gene inserted in an engineered RNA virus: efficient expression in monocotyledonous plant cells. Science 231, 1294–1297.PubMedGoogle Scholar
  37. Furtado, A., Henry, R., Scott, K. and Meech, S. (2003) The promoter of the asi gene directs expression in the maternal tissues of the seed in transgenic barley. Plant Mol. Biol. 52, 787–799.PubMedGoogle Scholar
  38. Furtado, A. and Henry, R. J. (2005) The wheat Em promoter drives reporter gene expression in embryo and aleurone tissue of transgenic barley and rice. Plant Biotech. J. 3, 421–434.Google Scholar
  39. Goodchild, J. (2004) Oligonucleotide therapeutics: 25 years agrowing. Curr. Opin. Mol. Ther. 6, 120–128.PubMedGoogle Scholar
  40. Greene, E. A., Codomo, C. A., Taylor, N. E., Henikoff, J. G., Till, B. J., Reynolds, S. H., Enns, L. C., Burtner, C., Johnson, J. E., Odden, A. R., Comai, L. and Henikoff, S. (2003) Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis. Genetics 164, 731–740.PubMedGoogle Scholar
  41. Halterman, D., Zhou, F., Wei, F., Wise, R. P. and Schulze-Lefert, P. (2001) The MLA6 coiled-coil, NBS-LRR protein confers AvrMla6-dependent resistance specificity to Blumeria graminis f. sp. hordei in barley and wheat. The Plant J. 25, 335–348.Google Scholar
  42. Haupt, S., Duncan, G. H., Holzberg, S. and Oparka, K. J. (2001) Evidence for symplastic phloem unloading in sink leaves of barley. Plant Physiol. 125, 209–218.PubMedGoogle Scholar
  43. Hein, I., Barciszewska-Pacak, M., Hrubikova, K., Williamson, S., Dinesen, M., Soenderby, I. E., Sundar, S., Jarmolowski, A., Shirasu, K. and Lacomme, C. (2005) Virus-induced gene silencing-based functional characterization of genes associated with powdery mildew resistance in barley. Plant Physiol. 138, 2155–2164.PubMedGoogle Scholar
  44. Hema, M. and Kao, C. C. (2004) Template sequence near the initiation nucleotide can modulate brome mosaic virus RNA accumulation in plant protoplasts. J. Virol. 78, 1169–1180.PubMedGoogle Scholar
  45. Hensel, G., Valkov, V., Middlefell-Williams, J. and Kumlehn, J. (2008) Efficient generation of transgenic barley: the way forward to modulate plant-microbe interactions. J. Plant Physiol. 165, 71–82.PubMedGoogle Scholar
  46. Himmelbach, A., Zierold, U., Hensel, G., Riechen, J., Douchkov, D., Schweizer, P. and Kumlehn, J. (2007) A set of modular binary vectors for the transformation of cereals. Plant Physiol. 145, 1192–1200.PubMedGoogle Scholar
  47. Hodgdon, A. L., Marcus, A. H., Arenaz, P., Rosichan, J. L., Bogyo, T. P. and Nilan, R. A. (1981) Ontogeny of the barley plant as related to mutation expression and detection of pollen mutations. Environ. Health Perspect. 37, 5–7.PubMedGoogle Scholar
  48. Holme, I. B., Brinch-Pedersen, H., Lange, M. and Holm, P. B. (2006) Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules. Plant Cell Rep. 25, 1325–1335.PubMedGoogle Scholar
  49. Holzberg, S., Brosio, P., Gross, C. and Pogue, G. P. (2002) Barley stripe mosaic virus-induced gene silencing in a monocot plant. The Plant J. 30, 315–327.Google Scholar
  50. Horvath, H., Rostoks, N., Brueggeman, R., Steffensen, B., von Wettstein, D. and Kleinhofs, A. (2003) Genetically engineered stem rust resistance in barley using the Rpg1 gene. Proc. Natl. Acad. Sci. USA 100, 364–369.PubMedGoogle Scholar
  51. Hu, T., Metz, S., Chay, C., Zhou, H. P., Biest, N., Chen, G., Cheng, M., Feng, X., Radionenko, M., Lu, F. and Fry, J. (2003) Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Rep. 21, 1010–1019.PubMedGoogle Scholar
  52. Ihlow, A., Schweizer, P. and Seiffert, U. (2008) A high-throughput screening system for barley/powdery mildew interactions based on automated analysis of light micrographs. BMC Plant Biol. 8, 6.PubMedGoogle Scholar
  53. Jackson, S. A., Zhang, P., Chen, W. P., Phillips, R. L., Friebe, B., Muthukrishnan, S. and Gill, B. S. (2001) High-resolution structural analysis of biolistic transgene integration into the genome of wheat. Theor. Appl. Genet. 103, 56–62.Google Scholar
  54. Jaehne, A., Becker, D., Brettschneider, H. and Loerz, H. (1994) Regeneration of transgenic, microspore-derived, fertile barley. Theor. Appl. Genet. 89, 525–533.Google Scholar
  55. Johansen, E., Edwards, M. C. and Hampton, R. O. (1994) Seed transmission of viruses: current perspectives. Annu. Rev. Phytopathol. 32, 363–386.Google Scholar
  56. Johnson, J. A., Bragg, J. N., Lawrence, D. M. and Jackson, A. O. (2003) Sequence elements controlling expression of Barley stripe mosaic virus subgenomic RNAs in vivo. Virology 313, 66–80.PubMedGoogle Scholar
  57. Kanyuka, K., Druka, A., Caldwell, D. G., Tymon, A., McCallum, N., Waugh, R. and Adams, M. J. (2005). Evidence that the recessive bymovirus resistance locus rym4 in barley corresponds to the eukaryotic translation initiation factor 4E gene. Mol. Plant Path. 6, 449–458.Google Scholar
  58. Kluth, A., Sprunck, S., Becker, D., Loerz, H. and Luetticke, S. (2002) 5′ deletion of a gbss1 promoter region from wheat leads to changes in tissue and developmental specificities. Plant Mol. Biol. 49, 669–682.PubMedGoogle Scholar
  59. Koprek, T., McElroy, D., Louwerse, J., Williams-Carrier, R. and Lemaux, P. G. (2000) An efficient method for dispersing Ds elements in the barley genome as a tool for determining gene function. The Plant J. 24, 253–264.Google Scholar
  60. Kumlehn, J., Serazetdinova, L., Hensel, G., Becker, D. and Loerz, H. (2006) Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollen cultures with Agrobacterium tumefaciens. Plant Biotech. J. 4, 251–261.Google Scholar
  61. Lacomme, C., Hrubikova, K. and Hein, I. (2003) Enhancement of virus-induced gene silencing through viral-based production of inverted-repeats. The Plant J. 34, 543–553.Google Scholar
  62. Lamacchia, C., Shewry, P. R., Di Fonzo, N., Forsyth, J. L., Harris, N., Lazzeri, P. A., Napier, J. A., Halford, N. G. and Barcelo, P. (2001) Endosperm-specific activity of a storage protein gene promoter in transgenic wheat seed. J. Exp. Bot. 52, 243–250.PubMedGoogle Scholar
  63. Li, S. L. and Redei, G. P. (1969) Estimation of mutation rate in autogamous diploids. Radiation Botany 9, 125–131.Google Scholar
  64. Li, J. R., Zhao, W., Li, Q. Z., Ye, X. G., An, B. Q., Li, X. and Zhang, X. S. (2005) RNA silencing of Waxy gene results in low levels of amylose in the seeds of transgenic wheat (Triticum aestivum L.). Acta Genet. Sin. 32, 846–854.PubMedGoogle Scholar
  65. Loukoianov, A., Yan, L., Blechl, A., Sanchez, A. and Dubcovsky, J. (2005) Regulation of VRN-1 vernalization genes in normal and transgenic polyploid wheat. Plant Physiol. 138, 2364–2373.PubMedGoogle Scholar
  66. Marwick, C. (1998) First “antisense” drug will treat CMV retinitis. J. Am. Med. Assoc. 280, 871.Google Scholar
  67. Matthes, M., Singh, R., Cheah, S. C. and Karp, A. (2001) Variation in oil palm (Elaeis guineensis Jacq.) tissue culture-derived regenerants revealed by AFLPs with methylation-sensitive enzymes. Theor. Appl. Genet. 102, 971–979.Google Scholar
  68. Matthews, P. R., Wang, M. B., Waterhouse, P. M., Thornton, S., Fieg, S. J., Gubler, F. and Jacobsen, J. V. (2001) Marker gene elimination from transgenic barley, using co-transformation with adjacent ‘twin T-DNAs’ on a standard Agrobacterium transformation vector. Mol. Breed. 7, 195–202.Google Scholar
  69. McCallum, C. M., Comai, L., Greene, E. A. and Henikoff, S. (2000a) Targeted screening for induced mutations. Nat. Biotech. 18, 455–457.Google Scholar
  70. McCallum, C. M., Comai, L., Greene, E. A. and Henikoff, S. (2000b) Targeting induced local lesions in genomes (TILLING) for plant functional genomics. Plant Physiol. 123, 439–442.Google Scholar
  71. McElroy, D., Zhang, W. G., Cao, J. and Wu, R. (1990) Isolation of an efficient actin promoter for use in rice transformation. The Plant Cell 2, 163–171.PubMedGoogle Scholar
  72. McKinney, H. H. and Greeley, L. W. (1965) Biological characteristics of barley stripe mosaic virus strains and their evolution. USDA Tech. Bull. 1324.Google Scholar
  73. Moissiard, G. and Voinnet, O. (2006) RNA silencing of host transcripts by cauliflower mosaic virus requires coordinated action of the four Arabidopsis Dicer-like proteins. Proc. Natl. Acad. Sci. USA 103, 19593–19598.PubMedGoogle Scholar
  74. Moutinho, A., Camacho, L., Haley, A., Pais, M. S., Trewavas, A. and Malho, R. (2001) Antisense perturbation of protein function in living pollen tubes. Sex. Plant Reprod. 14, 101–104.Google Scholar
  75. Murray, F., Brettell, R., Matthews, P., Bishop, D. and Jacobsen, J. (2004) Comparison of Agrobacterium-mediated transformation of four barley cultivars using the GFP and GUS reporter genes. Plant Cell Rep. 22, 397–402.PubMedGoogle Scholar
  76. Murray, F., Matthews, P., Jacobsen, J. and Gubler, F. (2006) Increased expression of HvGAMYB in transgenic barley increases hydrolytic enzyme production by aleurone cells in response to gibberellin. J. Cereal Sci. 44, 317–322.Google Scholar
  77. Nielsen, K., Olsen, O. and Oliver, R. (1999) A transient expression system to assay putative antifungal genes on powdery mildew infected barley leaves. Physiol. Mol. Plant Pathol. 54, 1–12.Google Scholar
  78. Ng, P. C. and Henikoff, S. (2003) SIFT: predicting amino acid changes that affect protein function. Nucleic Acids Res. 31, 3812–3814.PubMedGoogle Scholar
  79. Oikawa, A., Rahman, A., Yamashita, T., Taira, H. and Kidou, S. I. (2007) Virus-induced gene silencing of P23k in barley leaf reveals morphological changes involved in secondary wall formation. J. Exp. Bot. 58, 2617–2625.PubMedGoogle Scholar
  80. Patel, M., Johnson, J. S., Brettell, R. I. S., Jacobsen, J. and Xue, G. P. (2000) Transgenic barley expressing a fungal xylanase gene in the endosperm of the developing grains. Mol. Breed. 6, 113–123.Google Scholar
  81. Perry, J. A., Wang, T. L., Welham, T. J., Gardner, S., Pike, J. M., Yoshida, S. and Parniske, M. (2004) A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus. Plant Physiol. 131, 866–871.Google Scholar
  82. Popelka, J. C. and Altpeter, F. (2003) Agrobacterium tumefaciens-mediated genetic transformation of rye (Secale cereale L.). Mol. Breed. 11, 203–211.Google Scholar
  83. Popelka, J. C., Xu, J. and Altpeter, F. (2003) Generation of rye plants with low copy number after biolistic gene transfer and production of instantly marker-free transgenic rye. Transgenic Res. 12, 587–596.PubMedGoogle Scholar
  84. Qui, P., Shandilya, H., D’Alessio, J. M., O’Connor, K., Durocher, J. and Gerard, G. F. (2004) Mutation detection using Surveyor nuclease. Biotechniques 36, 702.Google Scholar
  85. Radchuk, V., Borisjuk, L., Radchuk, R., Steinbiss, H. H., Rolletschek, H., Broeders, S. and Wobus, U. (2006) Jekyll encodes a novel protein involved in the sexual reproduction of barley. The Plant Cell 18, 1652–1666.PubMedGoogle Scholar
  86. Rasco-Gaunt, S., Riley, A., Cannell, M., Barcelo, P. and Lazzeri, P. A. (2001) Procedures allowing the transformation of a range of European elite wheat (Triticum aestivum L.) varieties via particle bombardment. J. Exp. Bot. 52, 865–874.PubMedGoogle Scholar
  87. Regina, A., Bird, A., Topping, D., Bowden, S., Freeman, J., Barsby, T., Kosar-Hashemi, B., Li, Z., Rahman, S. and Morell, M. (2006) High-amylose wheat generated by RNA interference improves indices of large-bowel health in rats. Proc. Natl. Acad. Sci. USA 103, 3546–3551.PubMedGoogle Scholar
  88. Ruiz, M. T., Voinnet, O. and Baulcombe, D. C. (1998) Initiation and maintenance of virus-induced gene silencing. The Plant Cell 10, 937–946.PubMedGoogle Scholar
  89. Salvo-Garrido, H., Travella, S., Bilham, L. J., Harwood, W. A. and Snape, J. W. (2004) The distribution of transgene insertion sites in barley determined by physical and genetic mapping. Genetics 167, 1371–1379.Google Scholar
  90. Schultheiss, H., Hensel, G., Imani, J., Broeders, S., Sonnewald, U., Kogel, K. H., Kumlehn, J. and Hueckelhoven, R. (2005) Ectopic expression of constitutively activated RACB small GTPase in barley enhances susceptibility to powdery mildew and abiotic stress. Plant Physiol. 139, 353–362.PubMedGoogle Scholar
  91. Schweizer, P., Pokorny, J., Schulze-Lefert, P. and Dudler, R. (2000) Technical advance. Double-stranded RNA interferes with gene function at the single-cell level in cereals. The Plant J. 24, 895–903.Google Scholar
  92. Scofield, S. R., Huang, L., Brandt, A. S. and Gill, B. S. (2005) Development of a virus-induced gene-silencing system for hexaploid wheat and its use in functional analysis of the Lr21-mediated leaf rust resistance pathway. Plant Physiol. 138, 2165–2173.PubMedGoogle Scholar
  93. Shirasu, K., Nielsen, K., Piffanelli, P., Oliver, R. and Schulze-Lefert, P. (1999) Cell-autonomous complementation of mlo resistance using a biolistic transient expression system. The Plant J. 17, 293–299.Google Scholar
  94. Simons, K. J., Fellers, J. P., Trick, H. N., Zhang, Z., Tai, Y., Gill, B. K. and Faris, J. D. (2006) Molecular characterization of the major wheat domestication gene Q. Genetics 172, 547–555.PubMedGoogle Scholar
  95. Singh, J., Zhang, S., Chen, C., Cooper, L., Bregitzer, P., Sturbaum, A., Hayes, P. M. and Lemaux, P. G. (2006) High-frequency Ds remobilization over multiple generations in barley facilitates gene tagging in large genome cereals. Plant Mol. Biol. 62, 937–950.PubMedGoogle Scholar
  96. Skadsen, R. W., Sathish, P., Federico, M. L., Abebe, T., Fu, J. and Kaeppler, H. F. (2002) Cloning of the promotor for a novel barley gene, Lem1, and its organ-specific promotion of Gfp expression in lemma and palea. Plant Mol. Biol. 49, 545–555.PubMedGoogle Scholar
  97. Slade, A. J., Fuerstenberg, S. I., Loeffler, D., Steine, M. N. and Facciotti, D. (2005) A reverse genetic, non-transgenic approach to wheat crop improvement by TILLING. Nature Biotechnology 23, 75–81.PubMedGoogle Scholar
  98. Stadler, L. J. (1928a) Mutations in barley induced by x-rays and radium. Science 68, 186–197.Google Scholar
  99. Stadler, L. J. (1928b) Genetic effects of X-rays in maize. Proc. Natl. Acad. Sci. USA 14, 69–75.Google Scholar
  100. Stahl, R., Horvath, H., Van Fleet, J., Voetz, M., von Wettstein, D. and Wolf, N. (2002) T-DNA integration into the barley genome from single and double cassette vectors. Proc. Natl. Acad. Sci. USA 99, 2146–2151.PubMedGoogle Scholar
  101. Stein, N., Perovic, D., Kumlehn, J., Pellic, B., Stracke, S., Streng, S., Ordon, F. and Graner, A. (2005) The eukaryotic translation initiation factor 4E confers multiallelic recessive bymovirus resistance in Hordeum vulgare L. The Plant J. 42, 912–922.Google Scholar
  102. Stoeger, E., Williams, S., Keen, D. and Christou, P. (1999) Constitutive versus seed specific expression in transgenic wheat: temporal and spatial control. Trans. Res. 8, 73–82.Google Scholar
  103. Sun, C., Hoglund, A. S., Olsson, H., Mangelsen, E. and Jansson, C. (2005) Antisense oligodeoxynucleotide inhibition as a potent strategy in plant biology: identification of SUSIBA2 as a transcriptional activator in plant sugar signalling. The Plant J. 44, 128–138.Google Scholar
  104. Sun, C., Ridderstrale, K., Hoglund, A. S., Larsson, L. G. and Jansson, C. (2007) Sweet delivery – sugar translocators as ports of entry for antisense oligodeoxynucleotides in plant cells. The Plant J. 52, 1192–1198.Google Scholar
  105. Suzuki, T., Eiguchi, M., Kumamaru, T., Satoh, H., Matsusaka, H., Moriguchi, K., Nagato, Y. and Kurata, N. (2008) MNU-induced mutant pools and high performance TILLING enable finding of any gene mutation in rice. Mol. Genet. Genomics 279, 213–223.PubMedGoogle Scholar
  106. Talamè, V., Bovina, R., Sanguineti, M. C., Tuberosa, R., Lundqvist, U. and Salvi, S. (2008) TILLMore, a resource for the discovery of chemically induced mutants in barley. Plant Biotechnol. J. 6, 477–485.PubMedGoogle Scholar
  107. Taylor, N. E. and Greene, E. A. (2003) PARSESNP: a tool for the analysis of nucleotide polymorphisms. Nucleic Acids Res. 31, 3808–3811.PubMedGoogle Scholar
  108. Thorneycroft, D., Hosein, F., Thangavelu, M., Clark, J., Vizir, I., Burrell, M. M. and Ainsworth, C. (2003) Characterization of a gene from chromosome 1B encoding the large subunit of ADP glucose pyrophosphorylase from wheat: evolutionary divergence and differential expression of Agp2 genes between leaves and developing endosperm. Plant Biotechnol. J. 1, 259–270.PubMedGoogle Scholar
  109. Tilahun, A., Skadsen, R., Patel, M. and Kaeppler, H. (2006) The Lem2 gene promoter of barley directs cell- and development-specific expression of gfp in transgenic plants. Plant Biotechnol. J. 4, 35–44.Google Scholar
  110. Till, B. J., Reynolds, S. H., Weil, C., Springer, N., Burtner, C., Young, K., Bowers, E., Codomo, C. A., Enns, L. C., Odden, A. R., Greene, E. A., Comai, L. and Henikoff, S. (2004) Discovery of induced point mutations in maize genes by TILLING. BMC Plant Biol. 4, 12.PubMedGoogle Scholar
  111. Tingay, S., McElroy, D., Kalla, R., Fieg, S., Wang, M., Thornton, S. and Brettell, R. (1997) Agrobacterium tumefaciens-mediated barley transformation. The Plant J. 11, 1369–1376.Google Scholar
  112. Tomita, N., Ogihara, T. and Morishita, R. (2003) Transcription factors as molecular targets: molecular mechanisms of decoy ODN and their design. Curr. Drug Targets 4, 603–608.PubMedGoogle Scholar
  113. Travella, S., Klimm, T. E. and Keller, B. (2006) RNA interference-based gene silencing as an efficient tool for functional genomics in hexaploid bread wheat. Plant Physiol. 142, 6–20.PubMedGoogle Scholar
  114. Tsutsumi, N., Kanayama, K. and Tano, S. (1992) Suppression of α-amylase gene expression by antisense oligodeoxynucleotide in barley cultured aleurone layers. Jpn. J. Genet. 67, 147–154.PubMedGoogle Scholar
  115. Vasil, V., Castillo, A. M., Fromm, M. E. and Vasil, I. K. (1992) Herbicide resistant fertile transgenic wheat plants obtained by microprojectile bombardment of regenerable embryogenic callus. Biotechnology 10, 667–674.Google Scholar
  116. Veena, J. H., Doerge, R. W. and Gelvin, S. (2003) Transfer of T-DNA and Vir proteins to plant cells by Agrobacterium tumefaciens induces expression of host genes involved in mediating transformation and suppresses host defense gene expression. The Plant J. 35, 219–236.Google Scholar
  117. Vickers, C. E., Xue, G. and Gresshoff, P. M. (2006) A novel cis-acting element, ESP, contributes to high-level endosperm-specific expression in an oat globulin promoter. Plant Mol. Biol. 62, 195–214.PubMedGoogle Scholar
  118. Wahlestedt, C., Salmi, P., Good, L., Kela, J., Johnsson, T., Hokfelt, T., Broberger, C., Porreca, F., Lai, J., Ren, K., Ossipov, M., Koshkin, A., Jakobsen, N., Skouv, J., Oerum, H., Jacobsen, M. H. and Wengel, J. (2000) Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proc. Natl. Acad. Sci. USA 97, 5633–5638.PubMedGoogle Scholar
  119. Wan, Y. and Lemaux, P. G. (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol. 104, 37–48.PubMedGoogle Scholar
  120. Wang, M. B., Abbott, D. C., Upadhyaya, N. M., Jacobsen, J. V. and Waterhouse, P. M. (2001) Agrobacterium tumefaciens-mediated transformation of an elite Australian barley cultivar with virus resistance and reporter genes. Aus. J. Plant Physiol. 28, 149–156.Google Scholar
  121. Waugh, R., Leader, D. J., McCallum, N. and Caldwell, D. (2006) Harvesting the potential of induced biological diversity. Trends Plant Sci. 11, 71–79.PubMedGoogle Scholar
  122. Weeks, J. T., Anderson, O. D. and Blechl, A. E. (1993) Rapid production of multiple independent lines of fertile transgenic wheat (Triticum aestivum). Plant Physiol. 102, 1077–1084.PubMedGoogle Scholar
  123. Wesley, S. V., Helliwell, C. A., Smith, N. A., Wang, M. B., Rouse, D. T., Liu, Q., Gooding, P. S., Singh, S. P., Abbott, D., Stoutjesdijk, P. A., Robinson, S. P., Gleave, A. P., Green, A. G. and Waterhouse, P. M. (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. The Plant J. 27, 581–590.Google Scholar
  124. Wiley, P. R., Tosi, P., Evrard, A., Lovegrove, A., Jones, H. D. and Shewry, P. R. (2007) Promoter analysis and immunolocalisation show that puroindoline genes are exclusively expressed in starchy endosperm cells of wheat grain. Plant Mol. Biol. 64, 125–136.PubMedGoogle Scholar
  125. Wu, H., McCormac, A. C., Elliott, M. C. and Chen, D. F. (1998) Agrobacterium-mediated stable transformation of cell suspension cultures of barley (Hordeum vulgare L.). Plant Cell Tissue Organ Cult. 54, 161–171.Google Scholar
  126. Wu, H., Sparks, C., Amoah, B. and Jones, H. D. (2003) Factors influencing successful Agrobacterium-mediated genetic transformation of wheat. Plant Cell Rep. 21, 659–668.PubMedGoogle Scholar
  127. Yan, L., Loukoianov, A., Blechl, A., Tranquilli, G., Ramakrishna, W., SanMiguel, P., Bennetzen, J. L., Echenique, V. and Dubcovsky, J. (2004) The wheat VRN2 gene is a flowering repressor down-regulated by vernalization. Science 303, 1640–1644.PubMedGoogle Scholar
  128. Yang, B., Wen, X., Kodali, N. S., Oleykowski, C. A., Miller, C. G., Kulinski, J., Besack, D., Yeung, J. A., Kowalski, D. and Yeung, A. T. (2000) Purification, cloning, and characterization of the CEL I nuclease. Biochemistry 39, 3533–3541.PubMedGoogle Scholar
  129. Yao, Q., Cong, L., Chang, J. L., Li, K. X., Yang G. X. and He, G. Y. (2006) Low copy number gene transfer and stable expression in a commercial wheat cultivar via particle bombardment. J. Exp. Bot. 57, 3737–3746.PubMedGoogle Scholar
  130. Zhang, S., Cho, M. J., Koprek, T., Yun, R., Bregitzer, P. and Lemaux, P. G. (1999) Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using in vitro shoot meristematic cultures derived from germinated seedlings. Plant Cell Rep. 18, 959–966.Google Scholar
  131. Zimny, J., Becker, D., Brettschneider, R. and Lorz, H. (1995) Fertile, transgenic Triticale (X Triticosecale Wittmack). Mol. Breed. 1, 155–164.Google Scholar

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© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Scottish Crop Research InstituteInvergowrieUK
  2. 2.Leibniz Institute of Plant Genetics and Crop Plant ResearchGaterslebenGermany

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