Most plants are deficient in some essential amino acids, vitamins and minerals but a balanced diet provides adequate quantities of all. Problems arise when the diet is restricted to a single plant source, which is often the case for both humans and domestic animals in developing countries (Graham and Gregorio, 2001). For example, cereal storage proteins are deficient in lysine (Lys) and threonine (Thr) while legumes lack the sulphur-containing amino acids methionine (Met) and cysteine (Cys). A diet solely comprising one of these protein sources will therefore be deficient for one or more essential amino acids. Milled cereal grains are deficient in several vitamins, the most important of which are vitamins A, E and folate. Humans can synthesize vitamin A if provided with the precursor molecule β-carotene. D-1-deoxyxylulose 5-phosphate (DXP) synthase (DXS) has been demonstrated to catalyze the rate-limiting step in the formation of plastid-derived isoprenoids such as β-carotene (Enfissi et al., 2005). The first committed step leading to carotenoid biosynthesis is the linking of two geranylgeranyl pyrophosphate (GGPP) molecules to form phytoene. In order to engineer the conversion of GGPP into β-carotene in cereal endosperm, expression of at least five enzymes is required: PSY (phytoene synthase), PDS (phytoene desaturase), ZDS (zetacarotene desaturase), CRTISO (carotenoid isomerase) and LYCB (lycopene β-cyclase) (Sandmann et al., 2006). Effort can be simplified by using a bacterial phytoene desaturase (CrtI) capable of conversion of phytoene into alltrans lycopene. Two enzymes, BCH (β-carotene hydroxylase) and LYCE (lycopene ε-cyclase), divert the pathway to non-provitamin A xanthophylls that are lower in provitamin A value. High activity of DXS, PSY and bacterial CrtI, and higher activity of LYCB, compared to LYCE, in combination with lowering activity of  the BCH enzyme, would lead to optimal accumulation of β-carotene in the cereal endosperm.

1 Introduction
Vitamin E encompasses a class of lipid antioxidants consisting of four forms each of tocopherol and tocotrienol that possess α-tocopherol activity (DellaPenna and Pogson, 2006). Many human diseases such as certain types of cancer and neurodegenerative and cardiovascular diseases are associated with insufficient intake of vitamin E. Because of its high economical value and importance for human nutrition, much effort has been dedicated to elucidating the tocopherol biosynthetic pathway in plants and and to identify rate-limiting steps by over-expression of candidate genes in transgenic plants (Kanwischer et al., 2005; DellaPenna and Pogson, 2006). High activity of HPPD and HPT1, VTE3 and γ-TMT enzymes in cereal endosperm, would lead to optimal accumulation of α-tocopherol.

Vitamin C (ascorbic acid, AsA) is required for cardiovascular function, immune cell development, connective tissue, and iron utilization (Chen et al., 2003). Humans cannot synthesize ascorbic acid. Consequently, vitamin C must be acquired from dietary sources, primarily from plants rich in AsA. Dehydroascorbate reductase (DHAR) allows plants to recycle dehydroascorbate, therefore recapturing AsA before it is lost.

Tetrahydrofolate and its derivatives (vitamin folates) are essential cofactors for one-carbon transfer reactions in all organisms. Similar to bacteria and yeasts, plants make folates de novo from pterin, ρ-aminobenzoic acid
(PABA), and glutamate moieties (Hanson and Gregory, 2002). In contrast, humans and other animals lack a complete folate synthesis pathway, thus they depend on plants as the source of dietary folates. Inadequate dietary
levels of folate can lead to megaloblastic anemia, birth defects, impaired cognitive development, and increased risk of cardiovascular disease and cancer (Hossain et al., 2004 and therein). Folates are synthesized de novo from
pterins and ρ-aminobezoate (PABA) by means of a multi-step pathway, whereas pterins and PABA are synthesized from GTP and chorismate, respectively. The reaction catalysed by plant GTP cyclohydrolase-1 (GCH) is a ratedetermining step in de novo pterin and folate biosynthesis in plants (Hossain et al., 2004).

Minerals such as iron (Fe), zinc (Zn) and selenium (Se) are essential to the body in small amounts because they are either components of enzymes or act as cofactors in controlling chemical reactions. Populations with
Fe deficiency risk are infants, children, adolescents and pregnant women. Zn deficiency may result in low psychomotor and mental development (children), poor pregnancy, poor immune function, tiredness, and retarded
growth. More then 30% people are Zn deficient (White and Broadley, 2005). Selenium is implicated in the protection of body tissues against oxidative stress, in the maintenance of defences against infection and in the
modulation of growth and development. Se also optimizes the biological functionality of Zn and Fe in humans and has anticarcinogenic properties.

2 Cloning of genes, transformation vectors, rice and corn transformation
We have cloned all genes shown below and made appropriate endosperm-specific or constitutive expression vectors for maize and rice transformation, as appropriate. Our strategy calls for the simultaneously enhancement
of vitamins (A, E, C and folate), essential amino acids (Lys and Met), and minerals (Fe, Se and Zn), in cereal endosperm tissue. Rice and corn transformation has been performed as described using direct DNA transfer and
co-transformation of all genes on separate plasmids, together with the appropriate selectable marker (Christou et al., 1991; Drakakaki et al., 2005). 

3 Status Quo of the project
The following genes have been cloned, transferred into expression vectors for either endosperm or constitutive expression as appropriate and transferred into maize and/or rice. Transgenic plants are being regenerated and
these are currently being analyzed at the DNA, mRNA and metabolite levels: Pro-vitamin A: D-1-deoxyxylulose 5-phosphate (DXP) synthase (dxs), Phytoene synthase (psy), Phytoene desaturase (crtI), Lycopene β-cyclase
(lycb), β-carotene hydroxylase (bch). Vitamin C: Dehydroascorbate reductase (dhar). Vitamin E: HPP dioxygenase (HPPD) (pds1), Tocopherol cyclase (sdx1/vte1), Homogentisate Phytylprenyltransferase (hpt1/vte2), MPBQ
methyltransferase (vte3), γ-Tocopherol methyltransferase (γ-TMT) (vte4). Folate: GTP cyclohydrolase-1 (folE). Iron: Iron regulated metal transporter (IRT1), Nicotianamine synthase 1 (NAS1), Nicotianamine aminotransferase
(NAAT-A), Iron-phytosiderophore transporter (HvYS1), Ferritin (Ferritin), Phytase (PHYA3). Zinc: Zn transporter (ZAT). Selenium: ATP sulfurylase (APS1). Lysine: Aspartate kinase (AK), Dihydrodipicolinate synthase (DHPS).
Methionine: Cystathionine γ-synthase (CGS).

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