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.

https://science.sciencemag.org/content/sci/287/5451/303.full.pdf

Engineering the Provitamin A (β-Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm

Plant compounds that are perceived by humans to have color are generally referred to as 'pigments'. Their varied structures and colors have long fascinated chemists and biologists, who have examined their chemical and physical properties, their mode of synthesis, and their physiological and ecological roles. Plant pigments also have a long history of use by humans. The major classes of plant pigments, with the exception of the chlorophylls, are reviewed here. Anthocyanins, a class of flavonoids derived ultimately from phenylalanine, are water-soluble, synthesized in the cytosol, and localized in vacuoles. They provide a wide range of colors ranging from orange/red to violet/blue. In addition to various modifications to their structures, their specific color also depends on co-pigments, metal ions and pH. They are widely distributed in the plant kingdom. The lipid-soluble, yellow-to-red carotenoids, a subclass of terpenoids, are also distributed ubiquitously in plants. They are synthesized in chloroplasts and are essential to the integrity of the photosynthetic apparatus. Betalains, also conferring yellow-to-red colors, are nitrogen-containing water-soluble compounds derived from tyrosine that are found only in a limited number of plant lineages. In contrast to anthocyanins and carotenoids, the biosynthetic pathway of betalains is only partially understood. All three classes of pigments act as visible signals to attract insects, birds and animals for pollination and seed dispersal. They also protect plants from damage caused by UV and visible light.

https://onlinelibrary.wiley.com/doi/epdf/10.1111/j.1365-313X.2008.03447.x

Biosynthesis of plant pigments: anthocyanins, betalains and carotenoids

Food and Agricultural Organization of the United Nations PA N I S F I A T G u id e in e s o n fo o d fo r tific atio n w th m ic r o n u tr ie n ts Interest in micronutrient malnutrition has increased greatly over the last few years. One of the main reasons is the realization that micronutrient malnutrition contributes substantially to the global burden of disease. Furthermore, although micronutrient malnutrition is more frequent and severe in the developing world and among disadvantaged populations, it also represents a public health problem in some industrialized countries. Measures to correct micronutrient deficiencies aim at ensuring consumption of a balanced diet that is adequate in every nutrient. Unfortunately, this is far from being achieved everywhere since it requires universal access to adequate food and appropriate dietary habits. Food fortification has the dual advantage of being able to deliver nutrients to large segments of the population without requiring radical changes in food consumption patterns.

Guidelines on food fortification with micronutrients

Chapter 1: Overview of Zinc Nutrition ............................................................................................................... S99 1.1 Biological functions of zinc ............................................................................................................................. S99 1.2 Tissue zinc distribution and reserves .............................................................................................................. S99 1.3 Zinc metabolism ........................................................................................................................................... S100 1.4 Importance of zinc for human health........................................................................................................... S101 1.5 Human zinc requirements............................................................................................................................. S105 1.5.1 Adult men ............................................................................................................................................. S106 1.5.2 Adult women......................................................................................................................................... S109 1.5.3 Children ................................................................................................................................................ S110 1.5.4 Pregnancy.............................................................................................................................................. S111 1.5.5 Lactation ............................................................................................................................................... S112 1.6 Dietary sources of zinc and suggested revisions of Recommended Daily Intakes .................................... S112 1.6.1 Dietary sources of zinc and factors affecting the proportion of zinc available for absorption ........ S112 1.6.2 Revised estimates of dietary requirements and recommended intakes for zinc ............................... S114 1.7 Zinc toxicity.................................................................................................................................................... S118 1.8 Causes of zinc deficiency and groups at high risk ....................................................................................... S121 1.9 Summary ........................................................................................................................................................ S123

International Zinc Nutrition Consultative Group (IZiNCG) technical document #1. Assessment of the risk of zinc deficiency in populations and options for its control.

The biosynthesis and nutritional uses of carotenoids.

Rice (Oryza sativa L.), the major food staple for more than two billion people, contains neither beta-carotene (provitamin A) nor C40 carotenoid precursors thereof in its endosperm. To improve the nutritional value of rice, genetic engineering was chosen as a means to introduce the ability to make beta-carotene into rice endosperm tissue. Investigation of the biochemical properties of immature rice endosperm using [14C]-labelled substrates revealed the presence of geranyl geranyl diphosphate, the C20 general isoprenoid precursor necessary for C40 carotenoid biosynthesis. Phytoene synthase, which condenses two molecules of geranyl geranyl diphosphate, is the first of four specific enzymes necessary for beta-carotene biosynthesis in plants. Therefore, the Japonica rice model variety Taipei 309 was transformed by microprojectile bombardment with a cDNA coding for phytoene synthase from daffodil (Narcissus pseudonarcissus) under the control of either a constitutive or an endosperm-specific promoter. In transgenic rice plants, the daffodil enzyme is active, as measured by the in vivo accumulation of phytoene in rice endosperm. Thus, it is demonstrated for the first time that it is in principle possible to engineer a critical step in provitamin A biosynthesis in a non-photosynthetic, carotenoid-lacking plant tissue. These results have important implications for long-term prospects of overcoming worldwide vitamin A deficiency.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1997.11051071.x

Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis

Gene transfer into intact cells was achieved by electroporating zygotic wheat embryos without any special pretreatment. Electroporation was tissue specific in so far as scutellum cells were found to be much more susceptible to gene transfer than other cell types of the embryo. The orientation of the embryos in the electroporation chamber also influenced the number of transformed scutellum cells; during electroporation, as in electrophoresis, the negatively charged plasmid DNA molecules seemed to move towards the positive electrode. Therefore, the embryos were arranged so that the scutella faced the negative electrode. The use of plasmids carrying either two chimeric anthocyanin regulatory genes or a chimeric gusA gene allowed clear identification of transformed cells in the scutellum. On some of the embryos, more than 100 transformed scutellum cells were found after electroporation with single electric pulses of 275 V/cm discharged from a 960-μF capacitor and with 100 μg DNA/ml electroporation buffer. Using the anthocyanin marker system, visibly transformed cells grew to produce red sectors.

Gene transfer by electroporation into intact scutellum cells of wheat embryos.

In the following sections, a series of step-by-step protocols for standard molecular techniques widely used in the characterization of transgenic plants and in gene expression studies with plant cells are compiled.

Standard Molecular Techniques for the Analysis of Transgenic Plants

For many years of genetic manipulation in plants, direct uptake of naked DNA by plant protoplasts has been the sole alternative to Agrobacterium tumefaciens-mediated gene transfer The first experiments demonstrating direct gene transfer included the delivery of isolated plasmid DNA to protoplasts of petunia and tobacco in the presence of poly-L-ornithine or polyethylene glycol (PEG) [1–4] During the following years, protoplast transformation mediated by PEG [5] or electroporation [6] was substantially simplified and their efficiency in model systems was increased by several orders of magnitude (reviewed by Paszkowski et al [7])

Andreas Klöti

Papers

Engineering the Provitamin A (β-Carotene) Biosynthetic Pathway into (Carotenoid-Free) Rice Endosperm

Transgenic rice (Oryza sativa) endosperm expressing daffodil (Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key intermediate of provitamin A biosynthesis

Gene transfer by electroporation into intact scutellum cells of wheat embryos.

Standard Molecular Techniques for the Analysis of Transgenic Plants

PEG-mediated direct gene transfer and electroporation