Combinatorial genetic transformation of cereals and the creation of metabolic libraries for the carotenoid pathway.

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.

[1]  L. Herrera-Estrella,et al.  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☆ , 1998 .

[2]  M. Davey,et al.  Carbohydrate and osmotic requirements for high-frequency plant regeneration from protoplast-derived colonies of indica and japonica rice varieties , 1997 .

[3]  P. Fraser,et al.  Understanding carotenoid metabolism as a necessity for genetic engineering of crop plants. , 2006, Metabolic engineering.

[4]  M. McMullen,et al.  Osmotic treatment enhances particle bombardment-mediated transient and stable transformation of maize , 2004, Plant Cell Reports.

[5]  Claire Halpin,et al.  Gene stacking in transgenic plants--the challenge for 21st century plant biotechnology. , 2005, Plant biotechnology journal.

[6]  P. Christou,et al.  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 , 1991, Bio/Technology.

[7]  P. Christou,et al.  The genetic manipulation of medicinal and aromatic plants , 2007, Plant Cell Reports.

[8]  B. Swedlund,et al.  Sorbitol as the Primary Carbon Source for the Growth of Embryogenic Callus of Maize , 1993, Plant physiology.

[9]  R. Twyman,et al.  Plant Transformation Technology: Particle Bombardment , 2004 .

[10]  J. Bennetzen,et al.  The y1 gene of maize codes for phytoene synthase. , 1996, Genetics.

[11]  P. Christou,et al.  When more is better: multigene engineering in plants. , 2010, Trends in plant science.

[12]  K. Eskridge,et al.  Effect of Sugars in Wheat Anther Culture Media , 1994 .

[13]  Jürgen Breitenbach,et al.  Transgenic multivitamin corn through biofortification of endosperm with three vitamins representing three distinct metabolic pathways , 2009, Proceedings of the National Academy of Sciences.

[14]  F. Skoog,et al.  A revised medium for the growth and bioassay with tobacco tissue culture , 1962 .

[15]  Jürgen Breitenbach,et al.  Combinatorial genetic transformation generates a library of metabolic phenotypes for the carotenoid pathway in maize , 2008, Proceedings of the National Academy of Sciences.

[16]  Eleanore T Wurtzel,et al.  Gene Duplication in the Carotenoid Biosynthetic Pathway Preceded Evolution of the Grasses1 , 2004, Plant Physiology.

[17]  C. Martínez,et al.  Androgenesis of highly recalcitrant rice genotypes with maltose and silver nitrate , 1995 .

[18]  I. Potrykus,et al.  Callus formation from cell culture protoplasts of corn (Zea mays L.) , 1979, Theoretical and Applied Genetics.

[19]  M. Davey,et al.  An improved procedure for plant regeneration from indica and japonica rice protoplasts , 1995, Plant Cell Reports.

[20]  P. Christou,et al.  Progress in plant metabolic engineering. , 2004, Current opinion in biotechnology.

[21]  P. Christou,et al.  An Efficient Rice Transformation System Utilizing Mature Seed-derived Explants and a Portable, Inexpensive Particle Bombardment Device , 1998, Transgenic Research.

[22]  A. Frígola,et al.  Identification and quantification of carotenoids including geometrical isomers in fruit and vegetable juices by liquid chromatography with ultraviolet-diode array detection. , 2004, Journal of agricultural and food chemistry.