Abstract
Experiments were conducted to produce transgenic barley plants following infection of immature embryos with Agrobacterium tumefaciens. Transformed callus was obtained using hygromycin resistance as a selectable marker and either green fluorescent protein (GFP) or β-glucuronidase (GUS) as a reporter. Significantly reduced plant transformation frequencies were obtained with the GFP gene compared to GUS. However, GFP proved to be an excellent reporter of early transformation events and was used to compare four barley cultivars for efficiency in two phases of transformation: the generation of stably transformed barley callus and the regeneration of plantlets from transformed callus. Transformed callus was generated at a high frequency (47–76%) in all four cultivars. Regeneration of transformed plantlets was also achieved for all four cultivars although the frequency was much higher for Golden Promise than for the other three genotypes, reiterating that genotype is an important determinant in the regenerative ability of barley. This study has demonstrated for the first time that Agrobacterium-mediated transformation can be used to transform the Australian cultivars Sloop and Chebec.
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References
Ahlandsberg S, Sathish P, Sun C, Jansson C (1999) Green fluorescent protein as a reporter system in the transformation of barley cultivars. Physiol Plant 107:194–200
Aizawa H, Fukui Y, Yahara I (1997) Live dynamics of Dictyostelium cofilin suggests a role in remodelling actin latticework into bundles. J Cell Sci 110:2333–2344
Barakat A, Carels N, Bernardi G (1997) The distribution of genes in the genomes of Gramineae. Proc Natl Acad Sci USA 94:6857–6861
Barakat A, Gallois P, Raynal M, Mestre-Ortega D, Sallaud C, Guiderdoni E, Delseny M, Bernardi G (2000) The distribution of T-DNA in the genomes of transgenic Arabidopsis and rice. FEBS Lett 471:161–164
Barro F, Martin A, Lazzeri PA, Barceló P (1999) Medium optimisation for efficient somatic embryogenesis and plant regeneration from immature inflorescences and immature scutella of elite cultivars of wheat, barley and tritordeum. Euphytica 108:161–167
Bevan M, Barnes WM, Chilton M (1983) Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res 11:369–385
Bregitzer P, Dahleen LS, Campbell RD (1998) Enhancement of plant regeneration from embryogenic callus of commercial barley cultivars. Plant Cell Rep 17:941–945
Carlson AR, Letarte J, Chen J, Kasha KJ (2001) Visual screening of microspore-derived transgenic barley (Hordeum vulgare L.) with green fluorescent protein. Plant Cell Rep 20:331–337
Chiu WL, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter for plants. Curr Biol 6:325–330
Cho MJ, Choi HW, Buchanan BB, Lemaux PG (1999) Inheritance of tissue-specific expression of barley hordein promoter-uidA fusions in transgenic barley plants. Theor Appl Genet 98:1253–1262
Christensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin genes: genes, structures, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18:675–689
Elliott AR, Campbell JA, Brettell RIS, Grof CPL (1998) Agrobacterium-mediated transformation of sugarcane using GFP as a screenable marker. Aust J Plant Physiol 25:739–743
Elliott AR, Campbell JA, Dugdale B, Brettell RIS, Grof CPL (1999) Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells. Plant Cell Rep 18:707–714
Fang YD, Akula C, 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::barley genomic DNA junctions. J Plant Physiol 159:1131–1138
Garvin DF, Miller-Garvin JE, Viccars EA, Jacobsen JV, Brown AHD (1998) Identification of molecular markers linked to ant28–484, a mutation that eliminates proanthocyanidin production in barley seeds. Crop Sci 38:1250–1255
Haseloff J, Siemering KR (1998) The uses of GFP in plants. In: Chalfie M, Kain S (eds) Green fluorescent protein: strategies, applications and protocols. Wiley, New York, pp191–220
Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282
Horvath H, Huang JT, Wong O, Kohl E, Okita T, Kannangara CG, von Wettstein D (2000) The production of recombinant proteins in transgenic barley grains. Proc Natl Acad Sci USA 97:1914–1919
Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907
Jordan MC (2000) Green fluorescent protein as a visual marker for wheat transformation. Plant Cell Rep 19:1069–1075
Kaeppler HF, Menon GK, Skadsen RW, Nuutila AM, Carlson AR (2000) Transgenic oat plants via visual selection of cells expressing green fluorescent protein. Plant Cell Rep 19:661–666
Ke XY, McCormac AC, Harvey A, Lonsdale D, Chen DF, Elliott MC (2002) Manipulation of discriminatory T-DNA delivery by Agrobacterium into cells of immature embryos of barley and wheat. Euphytica 126:333–343
Kohli A, Gahakwa D, Vain P, Laurie DA, Christou P (1999) Transgene expression in rice engineered through particle bombardment: molecular factors controlling stable expression and transgene silencing. Planta 208:88–97
Kott LS, Howarth M, Peterson RL, Kasha KJ (1985) Light and electron microscopy of callus initiation from haploid barley embryos. Can J Bot 63:1801–1805
Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9:963–967
Lührs R, Lörz H (1987) Plant regeneration in vitro from embryogenic cultures of spring- and winter-type barley (Hordeum vulgare L.) varieties. Theor Appl Genet 75:16–25
Matthews PR, Wang MB, Waterhouse PM, Thornton S, Fieg SJ, Gubler F, Jacobsen JV (2001) Marker gene elimination from transgenic barley, using co-transformation with adjacent ‘twinT-DNAs’ on a standard Agrobacterium transformation vector. Mol Breed 7:195–202
McCormac AC, Wu H, Bao M, Wang Y, Xu R, Elliott MC, Chen DF (1998) The use of visual marker genes as cell-specific reporters of Agrobacterium-mediated T-DNA delivery to wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) Euphytica 99:17–25
Ohta S, Mita S, Hattori T, Nakamura K (1990) Construction and expression in tobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol 31:805–813
Patel M, Johnson JS, Brettell RIS, Jacobsen J, Xue GP (2000) Transgenic barley expressing a fungal xylanase gene in the endosperm of the developing grains. Mol Breed 6:113–123
Pesole G, Liuni S, Grillo G, Saccone C (1998) UTRdb: a specialized database of 5′- and 3′-untranslated regions of eukaryotic mRNAs. Nucleic Acids Res 26:192–195
Ryschka S, Ryschka U, Schulze J (1991) Anatomical studies on the development of somatic embryoids in wheat and barley explants. Biochem Physiol Pflanz 187:31–41
Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S, Brettell R (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J 11:1369–1376.
Trifonova A, Madsen S, Olesen A (2001) Agrobacterium-mediated transgene delivery and integration into barley under a range of in vitro culture conditions. Plant Sci 161:871–880
Van der Geest AHM, Petolino JF (1998) Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny. Plant Cell Rep 17:760–764
Walmsley AM, Henry RJ, Birch RG (1995) Optimisation of tissue culture conditions for transformation studies using immature embryos of Australian barley cultivars. Aust J Bot 43:499–504
Wan Y, Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104:37–48
Wang MB, Upadhyaya NM, Brettell RIS, Waterhouse PM (1997) Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens. J Gen Breed 51:325–334
Wang MB, Li Z, Matthews PR, Upadhyaya NM, Waterhouse PM (1998) Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants. Acta Hortic 461:401–407
Wang MB, Abbott DC, Upadhyaya NM, Jacobsen JV, Waterhouse PM (2001) Agrobacterium tumefaciens-mediated transformation of an elite Australian barley cultivar with virus resistance and reporter genes. Aust J Plant Physiol 28:149–156
Weir B, Gu X, Wang M, Upadhyaya N, Elliott AR, Brettell RIS (2001) Agrobacterium tumefaciens-mediated transformation of wheat using suspension cells as model system and green fluorescent protein as a visual marker. Aust J Plant Physiol 28:807–818
Acknowledgements
We thank Dr. J. Sheen (Harvard Medical School) for the gift of the sGFP(S65T) gene. The support of the Grains Research and Development Corporation (Australia) is gratefully acknowledged.
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Communicated by W. Harwood
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Murray, F., Brettell, R., Matthews, P. et al. Comparison of Agrobacterium-mediated transformation of four barley cultivars using the GFP and GUS reporter genes. Plant Cell Rep 22, 397–402 (2004). https://doi.org/10.1007/s00299-003-0704-8
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DOI: https://doi.org/10.1007/s00299-003-0704-8