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

Department of Plant Production and Forestry Science, ETSEA, University of Lleida, Lleida, Spain.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2012; 847:419-35. DOI: 10.1007/978-1-61779-558-9_33
Source: PubMed


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.

Download full-text


Available from: Changfu Zhu, Mar 12, 2014
1 Follower
126 Reads
  • Source
    • "Circular plasmids are then constructed by its endogenous homologous recombination machinery. With a similar mechanism, DNA assembly was also performed in other organisms such as Bacillus subtilis (Yonemura et al., 2007; Itaya et al., 2008) and certain plants (Zhu et al., 2008; Farre et al., 2012). Recently, a promising direct DNA cloning method in E. coli was reported by Fu et al. (2012; Zhang et al., 2000). "
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA assembly is one of the most important foundational technologies for synthetic biology and metabolic engineering. Since the development of the restriction digestion and ligation method in the early 1970s, a significant amount of effort has been devoted to developing better DNA assembly methods with higher efficiency, fidelity, and modularity, as well as simpler and faster protocols. This review will not only summarize the key DNA assembly methods and their recent applications, but also highlight the innovations in assembly schemes and the challenges in automating the DNA assembly methods. This article is protected by copyright. All rights reserved.
    FEMS Yeast Research 06/2014; DOI:10.1111/1567-1364.12171 · 2.82 Impact Factor
  • Source
    • "All transformation constructs are based on pUC8 plasmids. Their maps are shown in Fig. 5. Transgenic coloured calli were selected and cultured on MS selection medium (Farre et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Rice endosperm is devoid of carotenoids because the initial biosynthetic steps are absent. The early carotenogenesis reactions were constituted through co-transformation of endosperm-derived rice callus with phytoene synthase and phytoene desaturase transgenes. Subsequent steps in the pathway such as cyclization and hydroxylation reactions were catalyzed by endogenous rice enzymes in the endosperm. The carotenoid pathway was extended further by including a bacterial ketolase gene able to form astaxanthin, a high value carotenoid which is not a typical plant carotenoid. In addition to astaxanthin and precursors, a carotenoid accumulated in the transgenic callus which did not fit into the pathway to astaxanthin. This was subsequently identified as 4-keto-α-carotene by HPLC co-chromatography, chemical modification, mass spectrometry and the reconstruction of its biosynthesis pathway in Escherichia coli. We postulate that this keto carotenoid is formed from α-carotene which accumulates by combined reactions of the heterologous gene products and endogenous rice endosperm cyclization reactions.
    Phytochemistry 01/2014; 98. DOI:10.1016/j.phytochem.2013.12.008 · 2.55 Impact Factor
  • Source
    • "Plant synthetic biology has the potential of bringing about a significant impact on crop production. Engineering enhanced abiotic stress tolerance for growth in marginal lands, turning C3 plants into C4 plants (Caemmerer et al., 2012), constructing whole-organism biosensors or sentinels (Antunes et al., 2011), engineering highly challenging metabolic routes (Farre et al., 2012), and combinations of these are just some examples of high-impact goals within the reach of biotechnologists . Also, it has not escaped our notice that the proposed grammar can be easily adopted by other nonplant systems as well. "
    Planta Medica 08/2013; 79(13). DOI:10.1055/s-0033-1351820 · 2.15 Impact Factor
Show more