Abstract
We report on a novel transformation procedure for barley by Agrobacterium infection of in vitro cultured ovules. Ovules of the cultivar Golden Promise were isolated a few hours after pollination and infected with the Agrobacterium tumefaciens strain AGL0 carrying the binary vector pVec8-GFP. The vector harboured a hygromycin resistance gene and the green fluorescence protein (GFP) gene. GFP-expressing embryos were isolated from the ovules, regenerated to plants and investigated by Southern blot analysis. Transformation frequencies amounted to 3.1% with hygromycin selection and 0.8% without selection. Mendelian inheritance and stable expression of the GFP gene was confirmed in 18 independent lines over two generations. We conclude that the described technique allows for the rapid and direct generation of high quality transgenic plants.
Similar content being viewed by others
Abbreviations
- BAP:
-
Benzylaminopurine
- GFP:
-
Green fluorescence protein
- GFP+ embryo:
-
Embryo expressing GFP
- GFP− embryo:
-
Embryo not expressing GFP
- nos:
-
Nopaline synthetase
References
Bechtold N, Ellis J, Pelletier G (1993) In planta Agrobacterium mediated gene-transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci Paris, Life Sci 316:1194–1199
Bechtold N, Jaudeau B, Jolivet S, Maba B, Vezon D, Voisin R, Pelletier G (2000) The maternal chromosome set is the target of the T-DNA in the in planta transformation of Arabidopsis thaliana. Genetics 155:1875–1887
Bennett MD, Smith JB, Barclay I (1975) Early seed development in Triticeae. Phios T Roy Soc B 272:199–226
Bent AF (2000) Arabidopsis in planta transformation. Uses, mechanisms, and prospects for transformation of other species. Plant Physiol 124:1540–1547
Bregitzer P, Halbert SE, Lemaux PG (1998) Somaclonal variation in the progeny of transgenic barley. Theor Appl Genet 96:421–425. DOI: 10.1007/s001220050758
Bregitzer P, Tonks D (2003) Inheritance and expression of transgenes in barley. Crop Sci 43:4–12
Brinch-Pedersen H, Olesen A, Rasmussen SK, Holm PB (2000) Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytase. Mol Breed 6:195–206. DOI: 10.1023/A:1009690730620
Cheng M, Lowe BA, Spencer TM, Ye XD, Armstrong CL (2004) Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cell Dev-Plant 40:31–45. DOI: 10.1079/IVP2003501
Cho MJ, Jiang W, Lemaux PG (1998) Transformation of recalcitrant barley cultivars through improvement of regenerability and decreased albinism. Plant Sci 138:229–244. DOI: 10.1016/S0165-1781(98)00122-X
Choi HW, Lemaux PG, Cho MJ (2000) Increased chromosomal variation in transgenic versus nontransgenic barley (Hordeum vulgare L.) plants. Crop Sci 40:524–533
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743. DOI: 10.1046/j.1365-313x.1998.00343.x
Curtis IS, Nam HG (2001) Transgenic radish (Raphanus sativus L. longipinnatus Bailey) by floral-dip method–plant development and surfactant are important in optimizing transformation efficiency. Transgenic Res 10:363–371. DOI: 10.1023/A:1016600517293
Engell K (1989) Embryology of barley–time course and analysis of controlled fertilization and early embryo formation based on serial sections. Nord J Bot 9:265–280
Holm PB, Knudsen S, Mouritzen P, Negri D, Olsen FL, Roue C (1995) Regeneration of the barley zygote in ovule culture. Sex Plant Reprod 8:49–59
Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188. DOI: 10.1023/A:1006423110134
Kao KN, Michayluk MR (1975) Nutritional requirements for growth of Vicia hajastana cells and protoplasts at a very low population density in liquid media. Planta 126:105–110
Labra M, Vannini C, Grassi F, Bracale M, Balsemin M, Basso B, Sala F (2004) Genomic stability in Arabidopsis thaliana transgenic plants obtained by floral dip. Theor Appl Genet 109:1512–1518. DOI: 10.1007/s00122-004-1773-y
Lange M, Vincze E, Møller MG, Holm PB (2006) Molecular analysis of transgene and vector backbone integration into the barley genome following Agrobaterium-mediated transformation. Plant Cell Rep (in press)
Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Bio/Technol 9:963–967
Liu F, Cao MQ, Tao L, Li Y, Robaglia C, Tourneur C (1998) In planta transformation of Pakchoi (Brassica campestris L.) by infiltration of adult plants with Agrobacterium. Acta Hortic 467:187–193
Mantis NJ, Winans SC (1992) The Agrobacterium tumefaciens vir gene transcriptional activator virG is transcriptionally induced by acid pH and other stress stimuli. J Bacteriol 174:1189–1196
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 ‘twin T-DNAs’ on a standard Agrobacterium transformation vector. Mol Breed 7:195–202. DOI: 10.1023/A:1011333321893
Mogensen HL, Holm PB (1995) Dynamics of nuclear DNA quantities during zygote development in barley. Plant Cell 7:487–494
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497
Murray F, Brettell R, Matthews P, Bishop D, 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. DOI: 10.1007/s00299-003-0704-8
Narasimhulu SB, Deng X, Sarria R, Gelvin SB (1996) Early transcription of Agrobacterium T-DNA genes in tobacco and maize. Plant Cell 8:873–886
Srivastava V, Ow DW (2004) Marker-free site-specific gene integration in plants. Trends Biotechnol 22:627–629. DOI: 10.1016/j.tibtech.2004.10.002
Stachel SE, Messens E, Van Montagu M, Zambryski P (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–629
Stuitje AR, Verbree EC, van der Linden KH, Mietkiewska EM, Nap JP, Kneppers TJA (2003) Seed-expressed fluorescent proteins as versatile tools for easy (co)transformation and high-throughput functional genomics in Arabidopsis. Plant Biotechnol J 1:301–309. DOI: 10.1046/j.1467-7652.2003.00028.x
Sykes LC, Matthysse AG (1986) Time required for tumor induction by Agrobacterium tumefaciens. Appl Environ Microbiol 52:597–598
Tingay S, McElroy D. Kalla R, Fieg S, Wang MB, Thornton S, Brettell R (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J 11:1369–1376. DOI: 10.1046/j.1365-313X.1997.11061369.x
Travella S, Ross SM, Harden J, Everett C, Snape JW, Harwood WA (2005) A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep 23:780–789. DOI: 10.1007/s00299-004-0892-x
Trieu AT, Burleigh SH, Kardailsky IV, Maldonado-Mendoza IE, Versaw WK, Blaylock LA, Shin H, Chiou TJ, Katagi H, Dewbre GR, Weigel D, Harrison MJ (2000) Transformation of Medicago truncatula via infiltration of seedlings or flowering plants with Agrobacterium. Plant J 22:531–541. DOI: 10.1046/j.1365-313x.2000.00757.x
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. DOI: 10.1016/S0168-9452(01)00479-4
Virts EL, Gelvin SB (1985) Analysis of transfer of tumor-inducing plasmids from Agrobacterium tumefaciens to Petunia protoplasts. J Bacteriol 16:1030–1038
Wang WC, Menon G, Hansen G (2003) Development of a novel Agrobacterium-mediated transformation method to recover transgenic Brassica napus plants. Plant Cell Rep 22:274–281. DOI: 10.1007/s00299-003-0691-9
Ye GN, Stone D, Pang SZ, Creely W, Gonzalez K, Hinchee M (1999) Arabidopsis ovule is the target for Agrobacterium in planta vacuum infiltration transformation. Plant J 19:249–257. DOI: 10.1046/j.1365-313X.1999.00520.x
Acknowledgements
The authors thank Ole Bråd Hansen and Lis Bagnkop Holte for skillful technical assistance. Michael Hansen, Department of Plant Biology, the Royal Veterinary and Agricultural University, Denmark, is thanked for performing the confocal microscopy. This work was funded by a grant from the Danish Research Council.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by W. Harwood
Rights and permissions
About this article
Cite this article
Holme, I.B., Brinch-Pedersen, H., Lange, M. et al. Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules. Plant Cell Rep 25, 1325–1335 (2006). https://doi.org/10.1007/s00299-006-0188-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-006-0188-4