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Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries, including highly populated areas of Asia, Africa, and Latin America. Recombinant DNA technology was used to improve its nutritional value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the endosperm.
Vitamin A deficiency causes symptoms ranging from night blindness to those of xerophthalmia and keratomalacia, leading to total blindness. In Southeast Asia, it is estimated that a quarter of a million children go blind each year because of this nutritional deficiency (1). Furthermore, vitamin A deficiency exacerbates afflictions such as diarrhea, respiratory diseases, and childhood diseases such as measles (2, 3). It is estimated that 124 million children worldwide are deficient in vitamin A (4) and that improved nutrition could prevent 1 million to 2 million deaths annually among children (3). Oral delivery of vitamin A is problematic (5, 6), mainly due to the lack of infrastructure, so alternatives are urgently required. Success might be found in supplementation of a major staple food, rice, with provitamin A. Because no rice cultivars produce this provitamin in the endosperm, recombinant technologies rather than conventional breeding are required.
Immature rice endosperm is capable of synthesizing the early intermediate geranylgeranyl diphosphate, which can be used to produce the uncolored carotene phytoene by expressing the enzyme phytoene synthase in rice endosperm (7). The synthesis of beta-carotene requires the complementation with three additional plant enzymes: phytoene desaturase and zeta-carotene desaturase, each catalyzing the introduction of two double bonds, and lycopene beta-cyclase, encoded by the Icy gene. To reduce the transformation effort, a bacterial carotene desaturase, capable of introducing all four double bonds required, can be used.
We used Agrobacterium-mediated transformation to introduce the entire beta-carotene biosynthetic pathway into rice endosperm in a single transformation effort with three vectors (Fig. 1) (8). The vector pB19hpc combines the sequences for a plant phytoene synthase (psy) originating from daffodil (9) (Narcissus pseudonarcissus; GenBank accession number X78814) with the...