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Transgenic Plants

Volume 847 of the series Methods in Molecular Biology pp 419-435

Date:

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

  • Gemma FarreAffiliated withDepartment of Plant Production and Forestry Science, ETSEA, University of Lleida
  • , Shaista NaqviAffiliated withDepartment of Plant Production and Forestry Science, ETSEA, University of Lleida
  • , Georgina SanahujaAffiliated withDepartment of Plant Production and Forestry Science, ETSEA, University of Lleida
  • , Chao BaiAffiliated withDepartment of Plant Production and Forestry Science, ETSEA, University of Lleida
  • , Uxue Zorrilla-LópezAffiliated withDepartment of Plant Production and Forestry Science, ETSEA, University of Lleida
  • , Sol M. RiveraAffiliated withDepartment of Chemistry, ETSEA, University of Lleida
  • , Ramon CanelaAffiliated withDepartment of Chemistry, ETSEA, University of Lleida
  • , Gerhard SandmanAffiliated withBiosynthesis Group, Molecular Biosciences, J.W. Goethe Universitaet
  • , Richard M. TwymanAffiliated withDepartment of Biological Sciences, University of Warwick
    • , Teresa CapellAffiliated withDepartment of Plant Production and Forestry Science, ETSEA, University of Lleida
    • , Changfu ZhuAffiliated withDepartment of Plant Production and Forestry Science, ETSEA, University of Lleida
    • , Paul ChristouAffiliated withDepartment of Plant Production and Forestry Science, ETSEA, University of LleidaInstitució Catalana de Reserca i Estudis Avançats Email author 

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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