Advertisement

Combinatorial Genetic Transformation of Cereals and the Creation of Metabolic Libraries for the Carotenoid Pathway

  • Gemma Farre
  • Shaista Naqvi
  • Georgina Sanahuja
  • Chao Bai
  • Uxue Zorrilla-López
  • Sol M. Rivera
  • Ramon Canela
  • Gerhard Sandman
  • Richard M. Twyman
  • Teresa Capell
  • Changfu Zhu
  • Paul Christou
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 847)

Abstract

Combinatorial nuclear transformation is used to generate populations of transgenic plants containing random selections from a collection of input transgenes. This is a useful approach because it provides the means to test different combinations of genes without the need for separate transformation experiments, allowing the comprehensive analysis of metabolic pathways and other genetic systems requiring the coordinated expression of multiple genes. The principle of combinatorial nuclear transformation is demonstrated in this chapter through protocols developed in our laboratory that allow combinations of genes encoding enzymes in the carotenoid biosynthesis pathway to be introduced into rice and a white-endosperm variety of corn. These allow the accumulation of carotenoids to be screened initially by the colour of the endosperm, which ranges from white through various shades of yellow and orange depending on the types and quantities of carotenoids present. The protocols cover the preparation of DNA-coated metal particles, the transformation of corn and rice plants by particle bombardment, the regeneration of transgenic plants, the extraction of carotenoids from plant tissues, and their analysis by high-performance liquid chromatography.

Key words

Combinatorial transformation Metabolic pathway Metabolic engineering Carotenoid pathway Particle bombardment Transgenic plants Metabolite profiling 

References

  1. 1.
    Zhu, C., Naqvi, S., Breitenbach, J., Sandmann, G., Christou, P., and Capell, T. (2008) Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize. Proc. Natl. Acad. Sci. USA 105, 18232–18237.PubMedCrossRefGoogle Scholar
  2. 2.
    Gómez-Galera, S., Pelacho, A. M., Gené, A., Capell, T., and Christou, P. (2007) The genetic manipulation of medicinal and aromatic plants. Plant Cell Rep. 26, 1689–1715.PubMedCrossRefGoogle Scholar
  3. 3.
    Capell, T. and Christou, P. (2004) Progress in plant metabolic engineering. Curr. Opin. Biotechnol. 15, 148–154.PubMedCrossRefGoogle Scholar
  4. 4.
    Sandmann, G., Römer, S., and Fraser, P. D. (2006) Understanding carotenoid metabolism as a necessity for genetic engineering of crop plants. Metabol. Eng. 8, 291–302.CrossRefGoogle Scholar
  5. 5.
    Naqvi, S., Farré, G., Sanahuja, G., Capell, T., Zhu, C., and Christou, P. (2010) When more is better: multigene engineering in plants. Trends Plant Sci. 15, 48–56.PubMedCrossRefGoogle Scholar
  6. 6.
    Halpin, C. (2005) Gene stacking in transgenic plants – the challenge for 21st century plant biotechnology. Plant Biotechnol. J. 3, 141–155.PubMedCrossRefGoogle Scholar
  7. 7.
    Buckner, B., San-Miguel, P., and Bennetzen, J. L. (1996) The y1 gene of maize codes for phytoene synthase. Genetics 143, 479–488.PubMedGoogle Scholar
  8. 8.
    Gallagher, G. E., Mattews, P. D., Li, F., and Wurtzel, E. T. (2004) Gene duplication in the carotenoid biosynthetic pathway preceded evolution of the grasses. Plant Physiol. 135, 1776–1783.PubMedCrossRefGoogle Scholar
  9. 9.
    Potrykus, I., Harms, C. T., and Lorz, H. (1979) Callus formation from cell culture protoplasts of corn (Zea mays L.). Theor. Appl. Genet. 54, 209–214.CrossRefGoogle Scholar
  10. 10.
    Christou, P., Ford, T., and Kofron, M. (1991) Production of transgenic rice (Oryza sativa L.) plants from agronomically important indica and japonica varieties via electric discharge particle acceleration of exogenous DNA into immature zygotic embryos. Bio/Technol. 9, 957–962.CrossRefGoogle Scholar
  11. 11.
    Murashige, T., and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15, 473–497.CrossRefGoogle Scholar
  12. 12.
    Cortés, C., Esteve, M. J., Frígola, A., and Torregrosa, F. (2004) Identification and quantification of carotenoids including geometrical isomers in fruit and vegetable juices by liquid chromatography with ultraviolet-diode array detection. J. Agric. Food Chem. 52, 2203–2212.PubMedCrossRefGoogle Scholar
  13. 13.
    Britton, G., Liaaen-Jensen, S., Pfander, H., Mercadante, A., and Egeland, E. (eds) (2004) Carotenoid Handbook. Birkhäuser, Basel.Google Scholar
  14. 14.
    Jain, R. K., Davey, M. R., Cocking, E. C., and Wu, R. (1997) Carbohydrate and osmotic requirements for high-frequency plant regeneration from protoplast-derived colonies of indica and japonica rice varieties. J. Exp. Bot. 48, 751–758.CrossRefGoogle Scholar
  15. 15.
    Sudhakar, D., Duc, L.T., Bong, B.B., Tinjuangjun, P., Maqbool, S. B., Valdez, M., Jefferson, R., and Christou, P. (1998) An efficient rice transformation system utilizing mature seed-derived explants and a portable, inexpensive particle bombardment device Transgenic Res. 7, 289–294.CrossRefGoogle Scholar
  16. 16.
    Valdez, M., Cabera-Ponce, J. L., Sudhakar, D., Herrera-Estrella, L., and Christou, P. (1998) Transgenic Central American, West African and Asian elite rice varieties resulting from particle bombardment of foreign DNA into mature seed-derived explants utilizing three different bombardment devices. Ann. Bot. 82, 795–801.CrossRefGoogle Scholar
  17. 17.
    Navarro-Alvarez, W., Baenziger, P. S., Eskridge, K. M., Shelton, D. R., Gustafson, V. D., and Hugo, M. (1994) Effect of sugars in wheat anther culture media. Plant Breed. 112, 53–62.CrossRefGoogle Scholar
  18. 18.
    Jain, R. K., Khehra, G. S., Lee, S.-H., Blackhall, N.W., Marchant, R., Davey, M. R., Power, J. B., Cocking, E. C., and Gosal, S. S. (1995) An improved procedure for plant regeneration from indica and japonica rice protoplasts. Plant Cell Rep. 14, 515–519.CrossRefGoogle Scholar
  19. 19.
    Lenrini, Z., Reyes, P., Martinez, C. P., and Roca, W. M. (1995) Androgenesis of highly recalcitrant rice genotypes with maltose and silver nitrate. Plant Sci. 110, 127–138.CrossRefGoogle Scholar
  20. 20.
    Naqvi, S., Zhu, C., Farre, G., Ramessar, K., Bassie, L., Breitenbach, J., Perez Conesa, D., Ros, G., Sandmann, G., Capell, T., and Christou, P. (2009) Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways. Proc. Natl. Acad. Sci. USA 106, 7762–7767.PubMedCrossRefGoogle Scholar
  21. 21.
    Twyman, R. M., and Christou, P. (2004) Plant transformation technology – particle bombardment, in Handbook of Plant Biotechnology (Christou, P., ed.), John Wiley & Sons, NY, pp. 263–289.Google Scholar
  22. 22.
    Vain, P., McMullen, M. D., and Finer, J. J. (1993) Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize. Plant Cell Rep. 12, 84–88.CrossRefGoogle Scholar
  23. 23.
    Swedlund, B., and Locy, R. D. (1993) Sorbitol as the primary carbon source for the growth of embryogenic callus of maize. Plant Physiol. 103, 1339–1346.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Gemma Farre
    • 1
  • Shaista Naqvi
    • 1
  • Georgina Sanahuja
    • 1
  • Chao Bai
    • 1
  • Uxue Zorrilla-López
    • 1
  • Sol M. Rivera
    • 2
  • Ramon Canela
    • 2
  • Gerhard Sandman
    • 3
  • Richard M. Twyman
    • 4
  • Teresa Capell
    • 1
  • Changfu Zhu
    • 1
  • Paul Christou
    • 1
    • 5
  1. 1.Department of Plant Production and Forestry Science, ETSEAUniversity of LleidaLleidaSpain
  2. 2.Department of Chemistry, ETSEAUniversity of LleidaLleidaSpain
  3. 3.Biosynthesis Group, Molecular BiosciencesJ.W. Goethe UniversitaetFrankfurtGermany
  4. 4.Department of Biological SciencesUniversity of WarwickCoventryUK
  5. 5.Institució Catalana de Reserca i Estudis AvançatsBarcelonaSpain

Personalised recommendations