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Immature pollen-derived doubled haploid formation in barley cv. Golden Promise as a tool for transgene recombination

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Abstract

Barley transformation mediated by Agrobacterium tumefaciens is routinely performed in a number of laboratories. However, elimination of selectable marker genes and formation of plants homozygous for the transgene via conventional segregation is laborious and time-consuming. Here we suggest a concept that includes the production of primary transgenic plants via infection of immature embryos with A. tumefaciens followed by androgenetic generation of a segregating population of entirely homozygous plants. Selectable marker-free, truebreeding plants carrying a single-opy transgene integrant may thus be efficiently and rapidly obtained. However, amenability to Agrobacterium-mediated transformation as well as androgenetic potential is genotype-dependent. Efficient genetic transformation by infection of immature embryos is so far confined to the spring type cultivar ‘Golden Promise’ which, however, turned out to be recalcitrant in pollen embryogenesis. To facilitate androgenetic generation of homozygous segregants from primary transformants, we have established a method for embryogenic pollen culture in cv. Golden Promise that includes conventional cold-treatment and subsequent preculture of immature pollen under starvation conditions prior to transfer to complete nutrient medium. Further we show that conditioning of the pollen culture medium by co-culture of immature wheat pistils as well as addition of pistil-preconditioned medium considerably support androgenetic development. Employment of the established method using immature pollen of primary transgenic plants demonstrates that selectable marker-free, true-breeding transgenic progeny can be rapidly obtained pursuing the concept proposed. The protocol presented will be useful in functional genomics as well as in molecular breeding approaches.

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References

  • Chen S., Li X., Liu X., Xu H., Meng K., Xiao G., Wei X., Wang F., Zhu Z. 2005. Green fluorescent protein as a vital elimination marker to easily screen marker-free transgenic progeny derived from plants co-transformed with a double T-DNA binary vector system. Plant Cell Rep., 23: 625–631.

    Article  PubMed  CAS  Google Scholar 

  • Fang Y.-D., 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.

    Article  CAS  Google Scholar 

  • Funatsuki H., Kuroda H., Kihara M., Lazzeri P.A., Mueller E., Loerz H., Kishinami I. 1995. Fertile transgenic barley generated by direct DNA transfer to protoplasts. Theor. Appl. Genet., 91, 707–712.

    Article  CAS  Google Scholar 

  • Gustafson V.D., Baenziger P.S., Wright M.S., Stroup W.W., Yen Y. 1995. Isolated wheat microspore culture. Plant Cell, Tiss. Org. Cult., 42: 207–213.

    Article  Google Scholar 

  • Hellens, R., Mullineaux, P., Klee H. 2000. A guide to Agrobacterium binary Ti vectors. Trends Plant Sci., 5, 446–451.

    Article  PubMed  CAS  Google Scholar 

  • Hensel G., Kumlehn J. 2004. Genetic transformation of barley (Hordeum vulgare L.) by co-culture of immature embryos with Agrobacteria. In: Transgenic crops of the world — Essential protocols, ed. by Curtis I.S., Kluwer Academic Publishers, Dordrecht: 35–45.

    Google Scholar 

  • Hohn B., Levy A.A., Puchta H. 2001. Elimination of selection markers from transgenic plants. Curr. Opinion Biotechnol., 12, 139–143.

    Article  CAS  Google Scholar 

  • Holm P.B., Olsen O., Schnorf M., Brinch-Pedersen H., Knudsen S. 2000. Transformation of barley by microinjection into isolated zygote protoplasts. Transgenic Res., 9, 21–32.

    Article  PubMed  CAS  Google Scholar 

  • Horvath H., Huang J.T., Wong O., Kohl E., Okita T., Kannangara C.G., von Wettstein D. 2000. The production of recombinant proteins in transgenic barley grains. Proc. Natl. Acad. Sci. USA, 97, 1914–1919.

    Article  PubMed  CAS  Google Scholar 

  • Horvath H., Rostoks N., Brueggemann R., Steffenson B., von Wettstein D. Kleinhofs A. 2003. Genetically engineered stem rust resistance in barley using the Rpg1 gene. Proc. Natl. Acad. Sci. USA, 100, 364–369.

    Article  PubMed  CAS  Google Scholar 

  • Hu T.C., Kasha K.J. 1997. Improvement of isolated microspore culture of wheat (Triticum aestivum L.) through ovary co-culture. Plant Cell Rep., 16, 520–525.

    Article  CAS  Google Scholar 

  • Hunter C.P. 1987. Plant generation method. European Patent Application EP 0245898 2, Bulletin 87/45.

  • Indrianto A., Heberle-Bors E., Touraev A. 1999. Assessment of various stresses and carbohydrates for their effect on the induction of embryogenesis in isolated wheat microspore culture. Plant Sci., 143: 71–79.

    Article  CAS  Google Scholar 

  • Jaehne A., Becker D., Brettschneider R., Loerz H. 1994. Regeneration of transgenic, microspore-derived, fertile barley. Theor. Appl. Genet., 89, 525–533.

    Google Scholar 

  • Koehler F., Wenzel G. 1985. Regeneration of isolated barley microspores in conditioned media and trials to characterizs the responsible factor. J. Plant Physiol., 121, 181–191.

    Google Scholar 

  • Komari T., Hiei Y., Saito Y., Murai N., Kumashiro T. 1996. Vectors carrying two separate T-DNAs for co-transformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J., 10, 165–174.

    Article  PubMed  CAS  Google Scholar 

  • Koprek T., Haensch R., Nerlich A., Mendel R.R., Schulze J. 1996. Fertile transgenic barley of different cultivars obtained by adjustment of bombardment conditions to tissue response. Plant Sci., 119, 79–91.

    Article  CAS  Google Scholar 

  • Kumlehn J., Loerz H. 1999. Monitoring sporophytic development of individual microspores of barley (Hordeum vulgare L.). In: Anther and Pollen: From Biology to Biotechnology. ed. by Clément C., Pacini E., Audran J.-C., Springer, Berlin Heidelberg New York: 183–190.

    Google Scholar 

  • Kumlehn J., Serazetdinova L., Hensel G., Becker D., Loerz H. 2006. Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollen cultures with Agrobacterium tumefaciens. Plant Biotechnol. J., D01:10.1111/j.1467-7652.2005.00178.k.

  • Lazo G.R., Stein P.A., Ludwig R.A. 1991. A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Bio/Technol., 9, 963–967.

    Article  CAS  Google Scholar 

  • Li H., Devaux P. 2001. Enhancement of microspore culture effiency of recalcitrant barley genotypes. Plant Cell Rep., 20, 475–481.

    Article  CAS  Google Scholar 

  • Li H., Devaux P. 2003. High frequency regeneration of barley doubled haploid plants from isolated microspore culture. Plant Sci., 164, 379–386.

    Article  CAS  Google Scholar 

  • Matthews P.R., Wang M.B., Waterhouse P.M., Thornton S., Fieg S.J., Gubler F., 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.

    Article  CAS  Google Scholar 

  • Mejza S.J., Morgant V., DiBona D., Wong J.R. 1993. Plant regeneration from isolated microspores of Triticum aestivum. Plant Cell Rep., 12, 149–153.

    Article  Google Scholar 

  • Miki B., McHugh S. 2004. Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J. Biotechnol., 107, 193–232.

    Article  PubMed  CAS  Google Scholar 

  • Mordhorst A.P., Loerz H. 1993. Embryogenesis and development of isolated barley (Hordeum vulgare L.) microspores are influenced by the amount and composition of nitrogen sources in culture media. J. Plant Physiol., 142, 485–492.

    CAS  Google Scholar 

  • 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.

    Article  PubMed  CAS  Google Scholar 

  • Olsen F.L., 1991. Isolation and cultivation of embryogenic microspores from barley (Hordeum vulgare L.). Hereditas, 115, 255–266.

    PubMed  CAS  Google Scholar 

  • Palotta M.A., Graham R.D., Langridge P., Sparrow D.H.B., Barker S.J. 2000. RFLP mapping of manganese efficiency in barley. Theor. Appl. Genet., 101, 1100–1108.

    Article  Google Scholar 

  • Patel M., Johnson J.S., Brettell R.I.S., Jacobsen J., Xue G.P. 2000. Transgenic barley expressing a fungal xylanase gene in the endosperm of the developing grains. Mol. Breed., 6, 113–123.

    Article  CAS  Google Scholar 

  • Sambrook J., Fritsch E.F., Maniatis T. 1989. Molecular Cloning: A Laboratory Manual, 2nd. Edition, Cold Spring Harbor Laboratory Press, New York, NY, USA.

    Google Scholar 

  • Schuenmann P.H.D., Coia G., Waterhouse P.M. 2002. Biopharming the SimpliREDTM HIV diagnostic reagent in barley, potato and tobacco. Mol. Breed., 9, 113–121.

    Article  CAS  Google Scholar 

  • Schultheiss H., Hensel G., Imani J., Broeders S., Sonnewald U., Kogel K.-H., Kumlehn J., Hueckelhoven R. 2005. Ectopic expression of constitutively activated RACB small GTPase in barley enhances susceptibility to powdery mildew and abiotic stress. Plant Physiol., DOI: 10.1104/pp.105.066613

  • Stahl R., Horvath H., Van Fleet J., Voetz M., von Wettstein D., Wolf N. 2002. T-DNA integration into the barley genome from single and double cassette vectors. Proc. Natl. Acad. Sci. USA, 99, 2146–2151.

    Article  PubMed  CAS  Google Scholar 

  • Stein N., Perovic D., Kumlehn J., Pellio B., Stracke S., Streng S., Ordon F., Graner A. 2005. The eukaryotic initiation factor of translation 4E confers multiallelic recessive bymovirus resistance in Hordeum vulgare L. Plant J., 42, 912–922

    Article  PubMed  CAS  Google Scholar 

  • Tingay S., McElroy D., Kalla R., Feig S., Wang M., Thornton S., Brettell R. 1997. Agrobacterium tumefaciens-mediated barley transformation. Plant J., 11, 1369–1376.

    Article  CAS  Google Scholar 

  • Touraev A., Indrianto I., Wratschko I., Vicente O., Heberle-Bors E. 1996. Efficient microspore embryogenesis in wheat (Triticum aestivum L.) induced by starvation at high temperature. Sex. Plant Reprod., 9, 209–215.

    Article  Google Scholar 

  • Travella S., Ross S.M., Harden J., Everett C., Snape J.W., Harwood W.A. 2005. A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep., 23, 780–789.

    Article  PubMed  CAS  Google Scholar 

  • 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., 162, 871–880.

    Article  Google Scholar 

  • Wan Y., and Lemaux P.G. 1994. Generation of large numbers of independently transformed fertile barley plants. Plant Physiol., 104, 37–48.

    PubMed  CAS  Google Scholar 

  • Wang M.B., Abbott D.C., Upadhyaya N.M., Jacobsen J.V., Waterhouse P.M. 2001. Agrobacterium tumefaciens-mediated transformation of an elite Australian barley cultivar with virus resistance and reporter genes. Austral. J. Plant Physiol., 28, 149–156.

    Google Scholar 

  • Vancanneyt G., Schmidt R., O’Connor-Sanchez A., Willmitzer L., Rocha-Sosa M. 1990. Construction of an intron-containing marker gene: Splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated transformation. Mol. Gen. Genet., 220, 245–250.

    Article  PubMed  CAS  Google Scholar 

  • von Wettstein D., Mikhaylenko G., Froseth J.A., Kannangara C.G. 2000. Improved barley broiler feed with transgenic malt containing heat stable (1,3–1,4)-beta-glucanase. Proc. Natl. Acad. Sci. USA, 97, 13512–13517.

    Article  Google Scholar 

  • Zhang S., Cho M.J., Koprek T., Yun R., Bregitzer P., Lemaux P.G. 1999. Genetic transformation of commercial cultivars of oat (Avena sativa L.) and barley (Hordeum vulgare L.) using shoot meristematic cultures derived from germinated seedlings. Plant Cell Rep., 18, 959–966.

    Article  CAS  Google Scholar 

  • Zheng M.Y., Liu W., Weng Y., Polle E. and Konzak C.F. 2001. Culture of freshly isolated wheat (Triticum aestivum L.) microspores treated with inducer chemicals. Plant Cell Rep., 20, 685–690.

    Article  CAS  Google Scholar 

  • Zheng M.Y., Weng Y., Liu W., Konzak C.F. 2002. The effect of ovary-conditioned medium on microspore embryogenesis in common wheat (Triticum aestivum L.). Plant Cell Rep., 20, 802–807.

    Article  CAS  Google Scholar 

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Author notes

  1. María-José Coronado

    Present address: Plant Development and Nuclear Organization Unit, Biological Research Centre (CIB), CSIC, C/ Ramiro de Maeztu no 9, 28040, Madrid, Spain

  2. Götz Hensel & Sylvia Broeders

    Present address: EU, JRC, Institute for Reference Materials and Measurements, RM Unit, Retieseweg 111, 2440, Geel, Belgium

Authors and Affiliations

  1. Institute of Plant Genetics and Crop Plant Research, Corrensstrasse 3, D-06466, Gatersleben, Germany

    María-José Coronado, Götz Hensel, Sylvia Broeders, Ingrid Otto & Jochen Kumlehn

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  1. María-José Coronado
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Coronado, MJ., Hensel, G., Broeders, S. et al. Immature pollen-derived doubled haploid formation in barley cv. Golden Promise as a tool for transgene recombination. Acta Physiol Plant 27, 591–599 (2005). https://doi.org/10.1007/s11738-005-0063-x

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