Revista Colombiana de Biotecnología ISSN: 0123-3475 [email protected] Universidad Nacional de Colombia Colombia

Mayer, Jorge E. Golden , Golden Crops, Golden Prospects Revista Colombiana de Biotecnología, vol. IX, núm. 1, julio, 2007, pp. 22-34 Universidad Nacional de Colombia Bogotá, Colombia

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Golden Rice, Golden Crops, Golden Prospects

Arroz Dorado, cultivos dorados, perspectivas doradas

Jorge E. Mayer*

SUMMARY is probably the best known second-generation—nutrition quality enhanced—transgenic crop, but several others are following suit. Golden Rice was developed to deal with the problem of deficiency (VAD), which affects millions of people all over the world, especially small children in deve- loping countries. VAD not only causes blindness and increased morbidity, but a hitherto almost ignored increase in mortality. While ongoing supplementation and fortification programs are helping to reduce the burden, there is a strong need to develop biofortified crops capable of sustainably providing various micronutrients to target populations. Green and progress in the biochemical understan- ding of the mechanisms of biosynthesis and accumulation pathways of micronutrients have not only paved the way to address VAD but also deficiency of the micronutrients iron, zinc, vitamin E, folate, and essential amino acids. Here I provide an overview of the progress and the political environment of the Golden Rice Project as well as the status of a number of related projects in biofortification. Key words: Biofortification, micronutrient deficiency, , , beta-, nutrigenomics

RESUMEN El Arroz Dorado es probablemente el cultivo transgénico de segunda generación –es decir, en lo rela- cionado con el mejoramiento de la calidad nutricional– mejor conocido en el mundo, aunque varios otros proyectos están siguiendo su ejemplo. El Arroz Dorado fue desarrollado para ayudar a resolver el problema de la deficiencia de vitamina A (DVA), la cual afecta a millones de personas a nivel mun- dial, especialmente a niños pequeños en los países en desarrollo. La DVA no sólo causa ceguera y un incremento de la susceptibilidad a diversas enfermedades, sino que también un hasta ahora ignorado aumento de la mortalidad. Mientras que programas de suplementación y fortificación están contribu- yendo a reducir la carga, urge la creación de cultivos biofortificados, capaces de suplir de manera sus- tentable los micronutrientes necesarios a las poblaciones blanco. La biotecnología verde, y el progreso en el entendimiento de las rutas metabólicas responsables de la biosíntesis y acumulación de los micro- nutrientes a nivel bioquímico han preparado el camino para no sólo solucionar el problema de la DVA, sino también el de la deficiencia de micronutrientes como hierro, zinc, vitamina E, folato y aminoácidos esenciales. En este documento se da una visión general del progreso del proyecto del Arroz Dorado, así como del estado de proyectos afines en biofortificación. Palabras clave: Biofortificación, deficiencia de micronutrientes, deficiencia de vitamina A, carotenoides, beta-caroteno, nutrigenómica.

Recibido: mayo 4 de 2007 Aceptado: mayo 25 de 2007.

* Ph. D., MIP Law, Trade Marks Attorney; Golden Rice Project Manager, Campus Technologies Freiburg, Germany. [email protected] 22 GOLDEN RICE, GOLDEN CROPS, GOLDEN PROSPECTS

BACKGROUND mentation, have diffi culties reaching remote rural populations, and are dependent on a continuous As estimated by the UN´s Food and Agriculture fl o w of funds. Organisation, almost 800 million people living in 98 developing countries did not get enough food Biofortifi cation is an alternative, sustainable to lead a normal, healthy and active life in 2000. approach. It is based on the development of crops Around eleven million children die of malnutrition capable of producing and accumulating the desired every year, whereby a vast majority of those deaths micronutrients in the edible portions of the . are linked to micronutrient defi ciencies (Jones et This approach offers an opportunity to attain a ál., 2003). Nutritional defi ciencies with the highest more inclusive coverage, especially of the poorest impact on morbidity and mortality are related to the sectors of society who depend primarily on agricul- lack of iron, zinc, iodine and vitamin A (Welch and ture for their livelihood. Biofortifi ed crops may be Graham, 2004). obtained through conventional breeding of geneti- cally improved basic staple crops. Genetically im- The Golden Rice Project was conceived to proved parental lines can be obtained in various alleviate the vitamin A defi ciency (VAD) problem, ways, including interbreeding with wild relatives, because of the magnitude of the problem and the mutagenesis, or . consequent potential impact that a successful inter- vention would have. Yearly, half a million people— VAD is prevalent among the poor who depend mainly children—become blind as a consequence mainly on starchy crops for their daily energy in- of VAD, fi fty percent of whom may die within a year take, because most starchy crops, such as rice, of becoming blind. VAD severely affects the immu- sorghum, cassava or bananas do not contain β-ca- ne system; hence it is involved in many of these rotene (provitamin A), which our body in turn can children’s deaths in the guise of multiple diseases, convert into vitamin A. Half of the world’s popula- like measles and malaria (Caulfi eld et ál., 2004). tion eats rice on a regular basis, and again, for half It has been calculated that simple measures, like of those rice is the main dietary energy source (Ta- breastfeeding, combined with vitamin A and zinc ble 1). The situation is similar for other staple crops supplementation, could save 25 percent of those like cassava and banana in Africa. Dependence affected children (UNICEF, 2003). on these crops as the predominant food sources, Various public and international programmes therefore, necessarily leads to VAD and other mi- for supplementation, industrial fortifi cation and cronutrient defi ciencies, most severely affecting diet diversifi cation have achieved substantial im- children and pregnant women. provements but have diffi culty in attaining full co- Staple crops rich in β-carotene could substan- verage of the affected population and, above all, tially reduce VAD. In rice and in some other crops sustainability. Supplementation requires the pro- this can only be achieved using genetic enginee- vision of the needed micronutrients in the form of ring, because the biosynthetic pathway in the edible capsules, for example, while industrial fortifi cation portions has been turned off as part of the plant’s involves the addition of micronutrients to centrally developmental program. processed foodstuffs, like the addition of vitamin A to oil or butter. Supplementation activities require In the case of rice, there are a number of red the involvement of ten thousands of people for the and brown varieties, but these do not contain β- distribution and complicated logistics to reach the carotene but other pigments. The unpolished rice target population. Supplementation campaigns are grain contains other valuable nutrients, like vitamin very costly in logistical terms, even though they are B and essential lipids. It is the latter that makes it often coupled to vaccination campaigns. In small usually necessary to polish the rice grain, as the li- countries, like Nepal or Ghana, the costs amount to pids may become rancid by oxidation during stora- approximately $2 million a year (MOST and USAID ge, thus affecting the taste and smell of the grain. Micronutrient Program, 2004). Campaigns should preferably be carried out at least twice every year. Green produce carotenoids in the lea- The reality is that on a global scale ca. 45 percent of ves and often in the fl owers, and are the basis of children do not get any supplementation (UNICEF, some of the attractive, natural orange, yellow, and 2003). Both approaches, fortifi cation and supple- red pigments. Among the carotenoids produced in 23 Rev. Colomb. Biotecnol. Vol. IX No. 1 Julio 2007 22-34

Table 1. Rice-based societies, vitamin A defi ciency plants is β-carotene, the best plant source of vita- status and intake from diet. Countries have been min A. β-Carotene is converted into (vitamin divided into two groups according to rice consumption A) in the body, by central cleavage by an oxygena- per capita, i.e. with an approximate consumption of se, yielding two molecules of retinal. Not all provita- 400 or 200 grams of rice per person per day. SVAD, min A sources are equally good sources of vitamin prevalence of subclinical vitamin A defi ciency in children A. α-Carotene, for example, only yields one mole- under the age of six; DCI, total daily caloric intake from rice; DVAI, dietary vitamin A intake. Data compiled from cule with vitamin A activity. Bioconversion effi cien- FAO, UNICEF and USDA databases. The recommended cy can vary, depending on the bioavailability of the daily allowance for children is 300 µg per day. provitamin. It can vary from a stoichiometric ratio of 6:1 for like to 24:1 for green leafy ve- getables, where carotenoids may be entangled in SVAD DCI DVAI complex matrices (Yeum and Russell, 2002). Ani- % % µg/d mal sources deliver directly vitamin A, rather than ca. 400 g/d the provitamin, leading to high bioavailability. The Bangladesh 28 71 55.6 most effi cient conversion ratio of 2:1 is obtained from β-carotene dissolved in oil. Cambodia 42 72 90.3 Indonesia 26 51 289 biosynthesis is often turned off in non-photosynthetic tissues, like in the rice grain. In Lao PDR 42 66 141 Golden Rice, the carotenoid biosynthetic pathway Myanmar 35 68 86.8 has been switched back on by transformation with Viet Nam 12 65 95.8 equivalents of the dormant (Ye et ál., 2000). This feat was made possible by the advancements ca. 200 g/d in green technology made over the last two Brunei n.a. 28 216 decades. Golden Rice is the result of a collabora- Burkina Faso 46 6 65.2 tion between the laboratories of Profs and , at the Federal Institute of Techno- 12 29 288 logy, Switzerland, and the University of Freiburg, Cuba n.a. 15 125 Germany, respectively (Ye et ál., 2000). Improved lines with higher β-carotene content were develo- Guinea 40 33 229 ped through collaboration with the Swiss agribu- Guinea-Bissau 31 42 163 siness (Paine et ál., 2005). The target Guyana n.a. 31 55.5 of this public-private partnership was to produce enough β-carotene to cover the recommended dai- 57 30 124 ly requirements of children and adults alike. Korea, DPR n.a. 34 191

Korea, Rep n.a. 30 260 THE MAKING OF GOLDEN RICE Madagascar 42 48 49.7 The 1980s saw the cornerstones for the generation Malaysia n.a. 30 330 of transgenic plants being laid. Similarly, assembly Nepal 33 38 144 of the carotenoid biosynthetic pathway puzzle re- 23 42 106 lied on extensive research carried out in many la- boratories. The generation of the fi rst Golden Rice Senegal 61 30 106 prototype took a concerted effort of seven years, Sierra Leone 47 45 331 from 1992 to 1999, by the collaborating institutions. Potrykus’ and Beyer’s ambitious goal was to re-en- Sri Lanka n.a. 39 34.9 gineer the biosynthetic machinery of the rice grain Suriname n.a. 27 63.6 to produce and accumulate a pigment only found in Thailand 22 44 114 green tissues and fl ower petals, against the scep- ticism of many experts. Carotenogenic tissues not only have active metabolic pathways, including the presence of precursor molecules, but also depo- 24 GOLDEN RICE, GOLDEN CROPS, GOLDEN PROSPECTS sition mechanisms, e.g. carotenoid crystallization, khardt et ál., 1997). This indicates that at least one formation of oil droplets, sequestration of carote- desaturase was absent. Similarly, the expression noids into membranes or protein-lipid complexes. of carotene desaturase (CrtI) from the bacterium The non-carotenogenic starchy of rice Erwinia uredovora, which leads to the fully conju- is very low in lipids and apparently lacks means for gated β-carotene precursor molecule by carotenoid deposition. Another unknown in rice en- adding four double bonds, alone did not produce dosperm was the supply of precursor molecules. any colour in rice endosperm. As it turned out, the combination of these two genes led fi nally to the To make a long story short, Golden Rice tech- fi r st Golden Rice prototype in 1999 (Ye et ál., 2000) nology is based on the simple principle that not- (Figure 1). withstanding the fact that rice plants synthesise β-carotene in vegetative tissues but not in the Lycopene, a red pigment with an undecaene grain, most of the biosynthetic pathway chromophore, is an intermediate product of PSY are present in the grain. Addition of only two ge- and CRTI in transgenic plants, but is virtually absent nes was suffi cient to reconstitute the pathway and because it is utilised as a substrate by enzymes consequently to accumulate β-carotene in the en- down the pathway to generate α- and β-carotene dosperm. These facts were not at all clear in 1992, together with variable amounts of oxygenated ca- and some of the tools to detect low-level gene ex- rotenoids, such as lutein and zeaxanthin. The caro- pression were only being developed at the time. tenoid pattern observed in the endosperm revealed Hence, the genes responsible for β-carotene bio- that lycopene cyclases (LCY) and α- and β-caro- synthesis were introduced one by one, alone and in tene hydroxylases (HYD) are present in wild-type combination, into rice plants until the breakthrough rice endosperm, while PSY and one or both of the in 1999, when the fi rst Golden Rice prototype was plant carotene desaturases— desaturase fi n ally obtained (Ye et ál., 2000). Since then carote- (PDS) and ζ-carotene desaturase (ZDS)—are not noid production in various plant has been (Schaub et ál., 2005). manipulated either by increasing the production of precursor molecules or by blocking the synthesis of An alternative pathway activation mechanism, enzymes downstream of the desired product (Fra- observed in tomato (Lycopersicon esculentum) but ser and Bramley, 2004; Giuliano et ál., 2000). not in rice, is a feedback induction of endogenous carotenoid biosynthetic genes as a result of the Geranylgeranyl-diphosphate (GGDP) is a key presence of the . CRTI alone led to an intermediate in the biosynthesis of isoprenoids, a increase of β-carotene rather than lycopene, and substance class to which carotenoids also belong. endogenous carotenoid biosynthetic genes were GGDP and its precursor isopentenyl-diphosphate upregulated, except for PSY, which was repressed are synthesised from products of glycolysis. De- (Romer et ál., 2000). Such CRTI-dependent upre- rivatives of GGDP go through a number of iso- gulation may be based on the fact that the plant prenylation and cyclisation reactions, leading to desaturases, PDS and ZDS, together produce a number of important products, like tocopherols prolycopene, the tetra-cis confi gured form of lyco- (vitamin E), tocotrienols, carotenoids, abscisic acid pene (Bartley et ál., 1999), which is subsequently (a phytohormone involved in important regulatory isomerized to the trans form by lycopene cis-trans processes, like bud and seed dormancy, and va- isomerase (CRTISO), a recently identifi ed rious stress responses), gibberellic acid (another of this pathway (Isaacson et ál., 2004; Isaacson et phytohormone promoting growth and cell elonga- ál., 2002; Park et ál., 2002). In contrast, the bac- tion), chlorophyll and other components of the pho- terial CRTI leads to the exclusive formation of the tosynthetic machinery. all-trans form of lycopene. The above fi ndings are supported by increased formation of specifi c caro- As indicated above, genes were introduced tenogenic mRNAs and proteins in daffodil fl owers into rice one by one or in combination. Phytoene upon artifi cial accumulation of all-trans lycopene synthase (Psy) from daffodil ( pseudo- after inhibition of LCY, leading to elevated total ca- narcissus), which utilises GGDP to form phytoene, rotenoid content (Al-Babili et ál., 1999). a colourless carotene with a triene chromophore (Burkhardt et ál., 1997) alone led to phytoene ac- Wild-type rice endosperm displays low levels of cumulation but not to desaturated derivatives (Bur- all mRNAs required for xanthophyll formation, i.e. 25 Rev. Colomb. Biotecnol. Vol. IX No. 1 Julio 2007 22-34

Pyruvate + Glyceraldehyde-3-phosphate ↓ DXS 1-Deoxy-D-xylulose-5-phosphate ↓ ↓ Dimethylallyl-diphosphate + 3 Isopentenyl-diphosphate ↓ GGPPS Geranygeranyl-diphosphate ↓ PSY 15-15’-Phytoene ↓ ↓ PDS Vitamin E CRTI 9,9’-di-cis-ζ-Carotene ↓ ↓ ZDS 7,9,9’,7’-tetra-cis-Lycopene ↓ CRTISO All-trans-Lycopene β-LCY↓ α-LCY, β-LCY ↓ β-Carotene α-Carotene ↓α-HYD ↓ α-HYD, β-HYD Zeaxanthin Lutein

Figure 1. Carotene biosynthesis and re-engineering of the pathway in Golden Rice. DXS, 1-deoxy-D-xylulose-5- phosphate synthase; GGPPS, geranylgeranyl-diphosphate synthase; PSY, phytoene synthase; CRTI, bacterial carote- ne desaturase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; CRTISO, carotene cis-trans-isomerase; LCY, lycopene cyclase; HYD, carotene hydroxylase. In Golden Rice two transgenes have been introduced to reconstitute the complete pathway, all other enzymes are present and active in the rice grain. The two genes are Psy, from daffodil (Narcissus pseudonarcissus) in GR1 or (Zea mays) in GR2, and CrtI, from the soil bacterium Erwinia uredovora. CRTI replaces PDS, ZDS and CRTISO. Desaturation by CRTI procedes via all-trans intermediates, as opposed to the plant enzymes.

PSY, PDS, ZDS, CRTISO, β-LCY, ε-LCY, β-HYD, redox chain, employing quinones, a quinone reduc- and ε-HYD, although considering the sensitivity tase, and molecular oxygen as a terminal electron of the PCR method used, the PSY transcript was acceptor (Beyer et ál., 1989; Mayer et ál., 1990; effectively absent, which is consistent with the Nievelstein et ál., 1995) to which it is linked via an fact that introduction of PSY is required—but not oxidase (for a review see Kuntz, 2004). This redox suffi cient—to produce the golden phenotype. Also pathway is especially important in non-green ca- PDS and ZDS are probably expressed at too low rotenoid-bearing tissues like endosperm, while the levels to confer the golden phenotype. The en- photosynthetic electron transport is thought to play an analogous role in chloroplasts. Nevertheless, dosperm-specifi c expression of the PDS/ZDS sys- CRTI was the preferred over the plant- tem—instead of CRTI—in rice endosperm resulted own desaturases due to its multifunctionality and in the formation of comparable levels of coloured minimal requirements for co-factors. carotenoids. This shows that the rice endosperm provides the complex requirements for the activity It has been shown that, in contrast to PSY, CRTI of the plant desaturases. PDS requires namely a is not rate-limiting and is capable of desaturating lar- 26 GOLDEN RICE, GOLDEN CROPS, GOLDEN PROSPECTS ge amounts of phytoene, thereby increasing β-caro- provitamin A requirements of the target population. tene accumulation (Paine et ál., 2005). The primary Some rural populations in Bangladesh, for exam- sequence of CRTI is unrelated to the plant-type des- ple, eat more than 80 percent of their daily caloric aturases. This might explain its simpler co-factor intake in the form of rice. requirements and may therefore be more effective in rice endosperm than the plant-type desaturases. The fi rst improved Golden Rice, called GR1, On the other hand, CRTISO, which is also active in contained the phytoene synthase (Psy) gene from rice endosperm seems to have evolved from CRTI daffodil and the carotene desaturase (CrtI) gene (Isaacson et ál., 2002; Park et ál., 2002). from the bacterium Erwinia uredovora, as in the prototype Golden Rice. In GR1 both genes were Even though PSY, PDS, and ZDS expression in expressed only in the rice endosperm under the rice endosperm established the poly-cis pathway of control of the rice grain storage protein glutelin carotene desaturation, the β-carotene formed was , rather than constitutively as in the pro- predominantly in the all-trans form. Furthermore, totype (Figure 2). One reason for this modifi cation the endosperm from CRTI transgenic plants yiel- was to avoid any interference with light-harvesting ded the identical isomer ratio of β-, indi- capacity in leaves caused by a potential decrease cating that rice CRTISO determines the isomeric in lutein, as mentioned above (Al-Babili and Beyer, ratio (Isaacson et ál., 2004). The activity of CRTI- 2005). The levels of carotenoids obtained with GR1 SO is also responsible for the formation of cyclic in the fi eld were 6 µg/g on average, an almost four- end groups. In its absence, cyclic carotenoids and fold increase over the prototype. This β-carotenoid derived xanthophylls would not form in non-green content is expected to be able to cover the recom- tissues, as has been shown with the tangerine mended daily allowance (RDA) values for children mutation in tomato and in etioplasts from the if we factor in a modest intake of vegetables and Arabidopsis ccr2 mutant, both lacking functional fi s h or other animal sources, as is usually the case CRTISO (Isaacson et ál., 2002; Park et ál., 2002). in the rural diets of Southeast Asian countries.

The activity of rice the LCYs never proved to In complex biosynthetic pathways there is be rate limiting, since lycopene did not accumulate. usually a rate-limiting step, which in this case is Thus, yellow pigment formation in Golden Rice is PSY. Therefore, Psy genes from different plant the result of active intrinsic cyclases. No feedback sources were compared in separate transforma- regulatory loop seems to affect the expression of tion experiments. As it turned out, the maize and rice carotenoid biosynthetic genes present in rice, rice genes gave the best results when transfor- as indicated by the fact that all the corresponding med into rice, which led to the development of mRNA levels remained unchanged as compared to GR2, using the maize gene (Paine et ál., 2005). the wild-type. In the process, GR2 lines accumulating high amounts of carotenoids were obtained. One re- The fl ux of substrate into either branch of xan- gulatory clean line contained 37 µg/g carotenoid thophyll formation is controlled by the two LCYs, content, 80 percent of which was β-carotene. which convert lycopene into β-carotene (β,β-ca- This represents an astounding 23-fold increase rotene) and α-carotene (β, ε-carotene). However, over the prototype. the transcripts of the cyclases remained unchan- ged. This strongly indicates that the isomer ratio in The recommended daily allowance (RDA) of vi- lycopene determines the probability of β- or ε-ring tamin A for 1-3 year-old children is 300 µg (half the formation, in this case leading to a preferential for- RDA is enough to maintain vitamin A blood levels mation of β-carotene and the concomitant decrea- at a normal, healthy level) (Institute of Medicine, se in lutein. 2001). Based on a retinol equivalency ratio for β- carotene of 12:1, half the RDA would be provided in 72 g of GR2. This is perfectly compatible with rice PRODUCT DEVELOPMENT consumption levels in target countries, which lie at 100-200 g of rice per child per day. It was recognised that at 1.6 µg/g of β-carotene and in the absence of a more varied diet the GR The golden trait can in principle be directly en- prototype would not be able to fully cover the daily gineered into many different rice varieties, but be- 27 Rev. Colomb. Biotecnol. Vol. IX No. 1 Julio 2007 22-34

Figure 2. Gene construct used for the generation of Golden Rice. RB, T-DNA right border sequence; Glu, rice endosperm-specifi c glutelin promoter; tpSSU, pea ribulose bis-phosphate carboxylase small subunit transit peptide for chloroplast localisation; nos, nopaline synthase terminator; Psy, phytoene synthase gene from Narcissus pseudonar- cissus (GR1) or Zea mays (GR2); Ubi1, maize polyubiquitin promoter; Pmi, phosphomannose isomerase gene from E. coli for positive selection; LB, T-DNA left border sequence.

cause of the stringent regulatory requirements, the guidance to the project. At the next level, a network aim is to have only one regulatory clean event as of national institutions participates in the develop- the starter seed for introgression in multiple bree- ment and distribution of locally adapted varieties. ding programmes (Hoa et ál., 2003). To date, the Golden Rice Network includes 16 na- tional institutions in Bangladesh, China, India, Indo- nesia, Nepal, the Philippines, Vietnam, and South REACHING OUT Africa. The Network is under the strategic guidance Golden Rice will be made available to developing of the Golden Rice Humanitarian Board and under countries within the framework of a humanitarian the management of a network coordinator, based project. This was, from the onset, a public research at the International Rice Research Institute (IRRI), project designed to reduce malnutrition in develo- in the Philippines. ping countries. The hurdle of intellectual property If we put aside the research investment by Syn- rights attached to the technologies, mainly in the genta so far, investment in the public sector has form of patents (Kryder et ál., 2000) used in the been relatively modest thus far ($2.4 million over production of Golden Rice was overcome thanks nine years). Product development, however, is to strong support from the private sector in the time-consuming and requires substantial additional form of free licences for humanitarian use. This funding. Funds for product development are nor- arrangement opened the way to collaborations mally not provided by the public sector, since the with public rice research institutions in developing work involved is not academic in . In univer- countries, providing freedom to operate to deve- sities researchers are evaluated based on scientifi c lop locally adapted Golden Rice varieties. productivity, and not marketable products. The lat- The Golden Rice approach does not interfere ter is the realm of industry. with a continued use of traditional varieties. The gol- Expenses increase even more dramatically den trait can be bred into any local variety by con- when it comes to biosafety assessment as required ventional means within four to six back-crosses or for regulatory purposes. But once a novel, bioforti- two years of breeding with the help of marker-assis- fi e d variety has been approved and handed over to ted selection. The locally adapted golden varieties farmers, the system can develop its full potential. will become the property of the farmers and they will From this point on, the technology is built into each also be able to use part of their harvest to sow in and every seed and does not require additional in- the next growing season, without restrictions. Fur- vestment. thermore, Golden Rice is compatible with the use of traditional farming systems; it does not require more agronomic inputs than the parental variety. GOLDEN CROPS

Moving Golden Rice from the laboratory to the The Golden Rice achievement demonstrates that fi e ld is not a trivial exercise. A group of international sometimes even complex metabolic pathways can experts from reputed institutions provides strategic be reactivated or reconstructed, even in tissues 28 GOLDEN RICE, GOLDEN CROPS, GOLDEN PROSPECTS that are not expected to be able to accumulate In a collaboration between the Tata Energy the desired metabolites. This fi nding can also be Research Institute (TERI) and in India, of importance to researchers working on the pro- Indian mustard (Brassica juncea), a popular duction of plant-derived pharmaceuticals (Ma et ál., source of cooking oil in India, was transformed 2005) but in the context of the present review it is with bacterial Psy and CrtI genes using Monsanto relevant for those who want to improve the nutritio- technology developed for the transformation of nal quality of foodstuffs, especially with regards to canola (Brassica napus). The idea behind this enrichment with micronutrients. Some of the best approach is to exploit the high bioavailability of β- starchy caloric sources in the world lack adequa- carotene in oil as a means of supplying it to the te amounts of micronutrients, not only carotenoids target population in a highly effective carrier. This and other vitamins but also minerals and essential project is now awaiting an ex-ante socio-economic amino acids. Golden Rice has served as a proof assessment before going ahead (Agricultural of concept for biofortifi cation obtained through ge- Biotechnology Support Project, 2003). netic engineering and has thus inspired other pro- jects along the same vein. But while the concept Similarly, in an USAID funded project for Africa, as such is transferable, still each crop requires its Iowa State University is developing “Golden Maize” own fi x, depending on the presence or absence of (Zea mays) by introducing the bacterial CrtB active enzymes along the metabolic and catabolic (phytoene synthase) and CrtI (carotene desaturase) pathways involved. genes (Rodermel et ál., 2006). Lines accumulating 4-14 µg/g dry weight have been obtained, Potato is one of the major staple crops in the which meets the 50 percent estimated average world, but in most β-carotene is present requirement (EAR) of vitamin A recommended for only in trace amounts. While the wild potato species children by USAID and HarvestPlus. While in rice Solanum phureja can contain high levels of carote- options are restricted because the embryo is lost noids, only a small fraction consists of β-carotene. during polishing, in maize carotene deposition can This fact calls for a transgenic solution to improve be directed to the endosperm or the embryo. The the nutritional quality of this important crop. This feat latter option deals with the fact that there would be has been meanwhile accomplished with the partici- less of a problem with the adoption of yellow maize pation of Peter Beyer’s lab, one of the inventors of in regions where white varieties are preferred and Golden Rice. In one approach, increase of β-caro- also that the embryo has a higher oil concentration, tene in potato was obtained by blocking the expres- thus offering a favourable environment for sion of ε-lycopene cyclase (ε-LCY), which diverts carotenoids and making it also more bioavailable. lycopene into the α-carotene and lutein branch of the pathway, using a tuber-specifi c RNA-silencing In 2005 the Bill & Melinda Gates Foundation approach (Diretto et ál., 2006). Using this approach launched their Grand Challenges in Global Health β-carotene content increased up to 14-fold. Re- initiative. Out of the 44 projects being funded within markably, lutein levels were not reduced in these that initiative four are in the agricultural sector, and tubers, indicating that ε-LCY is not rate-limiting. An all are about crop biofortifi cation. The four projects even greater increase in carotenoid production in address the challenge to ‘Improve nutrition to pro- potato was obtained by introducing a mini-pathway mote health by creating a full range of optimal, bio- consisting of three genes from Erwinia: phytoene available nutrients in a single staple plant species.’ synthase (CrtB), phytoene desaturase (CrtI), and The gauntlet has been taken up by four internatio- lycopene β-cyclase (CrtY) (Diretto et ál., 2007). Total nal consortia, led by the Queensland University of carotenoid content increased 20-fold while β-caro- Technology for banana (Musa spp) (http://www.ihbi. tene increased 3600-fold, up to 47 µg/g dry weight. qut.edu.au/research/tropical/biofort/), Ohio State Unexpectedly, phytoene also stays high in these li- University for cassava (Manihot esculenta) (http:// nes, suggesting that desaturation is still rate limiting, www.biocassavaplus.org/), the Africa Biofortifi ed as opposed to canola, where expression of CrtI and Project for sorghum (Sorghum bicolor) (http://www. CrtB leaves only trace amounts of phytoene (Rava- supersorghum.org/), and the University of Freiburg nello et ál., 2003; Shewmaker et ál., 1999). Another for rice ( sativa) (http://www.goldenrice.org/ difference is that expression of CrtY does not lead to Content5-GCGH/GCGH1.html). The grant awar- increased production of total carotenoids. ded to the University of Freiburg is being utilized to 29 Rev. Colomb. Biotecnol. Vol. IX No. 1 Julio 2007 22-34 further improve Golden Rice by adding genes that tion of β-carotene to levels of up to 6 mg/g dry weight will promote the accumulation of iron, zinc, vitamin in root tissues, seems to be due to the segregation E and high-quality protein in rice grains. The other of a yet unknown dominant inhibitor (Buishand and consortia mentioned are pursuing similar goals in Gabelman, 1979). These mutants accumulate large addition to combinations with genetic traits for bio- amounts of β-carotene, although its bioavailability tic and abiotic stress resistance and adaptations. is rather low, because it is deposited in crystalline Recently, the re-engineering of the folate biosyn- form. Bioavailability can be raised by cooking and thetic pathway in tomato was reported (Diaz de la serving with some oil though (Jayarajan et ál., 1980; Garza et ál., 2007), and there are already ongoing Roodenburg et ál., 2000). Cooking generally has a discussions about including this trait into the crops positive effect on bioavailability, while retention of mentioned above. carotenoids after heat treatment is very high, as was shown for orange-fl eshed sweepotato (van Jaars- Again, for the establishment of β-carotene veld et ál., 2005). biosynthesis in these very different crops and tis- sues (grains, roots and fruits) it will be necessary A more recent discovery is the case of the al- to determine the presence or absence of biosyn- ready commercially available orange-coloured thetic, downstream and catabolic genes case by caulifl ower (Brassica oleracea var botrytis). This case. While cassava and banana varieties with semi-dominant mutation is due to the insertion of a high β-carotene content are available (Chaves et retrotransposon into the so-called Or allele (Or for ál., 2000; Englberger et ál., 2003; Englberger et ál., orange) (Lu et ál., 2006). Although its role is not 2006; Iglesias et ál., 1997), the problem in these completely understood, Or is known to be involved specifi c cases is that these crops are not amenable in proplastid differentiation and other non-coloured to breeding because of lack of sexuality in the case into chromoplasts for carotenoid accumula- of banana, and because of the agricultural use of tion. Thus, rather than exerting an infl uence on caro- vegetative propagation for the highly heterozygous tenoid biosynthesis, Or most likely acts by increasing cassava plants, for the purpose of genotype pre- sink strength and thus sequestration of carotenoids. servation. The principle is transferable, as was shown through the generation of yellow-fl eshed potatoes by expre- As mentioned above, all these crops constitute ssing Or in transgenic potatoes (Lu et ál., 2006). The the main caloric intake in many developing coun- downside of this approach thus far is that in homo- tries where rice is not the main food source, and zygous plants growth is stunted, probably due to not they all lack essential micronutrients. It is very en- well understood pleiotropic effects. couraging to see that the Golden Rice approach has played a catalytic role in inspiring these activi- ties, and it can only be hoped that people in need OUTLOOK will soon have access to better nutrition thanks to these projects and the support of people with a vi- There is plenty of goodwill in the public and in sion and understanding. the private sectors worldwide to exploit the po- tential of green biotechnology for the benefi t of the poor. However, without a realistic risk assess- NATURAL GAMES WITH COLOUR ment approach, funds for public research will not suffi ce to deliver on the promise of biotechnology. Sometimes, in nature, mutations lead to the acti- Under circumstances like the extreme use of the vation of carotenoid biosynthesis in a usually non- precautionary principle, scientifi c progress will be- carotenogenic organ. This was the case for carrots come detached from product development and the (Daucus carota), a plant originating in Afghanistan population at large would not benefi t from the great and brought initially to Europe in various colours ex- potential of the technology. cept for orange. Carrots containing purple-red antho- cyanins were fi rst grown in the Middle and Far East, Hopefully some countries affected by VAD will along with white, red, yellow, green and black versio- be able to release locally adapted Golden Rice ns. The Dutch then introduced the orange mutant in varieties within a couple of years, pending regu- honour of Dutch Royal House of Orange in the 16th latory approval. Considering that Golden Rice century. The mutation, which leads to the accumula- could substantially reduce morbidity and mortali- 30 GOLDEN RICE, GOLDEN CROPS, GOLDEN PROSPECTS ty, it is not easily understandable why the project from a number of socio-economic ex-ante studies is not getting wider international support to carry carried out to predict the impact of Golden Rice it through the regulatory process following the fast adoption in selected target countries. A recent track. Notwithstanding the fact that during the last World Bank report concluded that the potential 20 years a vast knowledge base on the produc- welfare improvement for Southeast Asian tion and commercialisation of genetically modifi ed countries, derived from the adoption of Golden plants has accumulated, a substantial amount of Rice, would be in the range of billions of dollars time and money will have to be spent on carrying annually (Anderson et ál., 2004). This gain in terms out the required biosafety assessments. Approval of health improvement of the population and the of a new line might cost in the range of $4 million economic benefi t to the country would dwarf the to comply with all requirements of toxicology and negative impact that the adoption of transgenic allergenicity testing, biochemical equivalency, envi- technology could have on trade because of import ronmental effects and other tests (Ramaswami and bans on transgenic commodities. As applicable Pray, 2007). to most developing countries, the largest share of the production of rice and most other crops is Golden Rice has already gone through many consumed locally (Binenbaum et ál., 2000). As it greenhouse tests and two fi eld trials that were car- turns our for India and Bangladesh, more than 50 ried out in Louisiana, because at the time target percent of deaths of children under fi ve years of countries had no biosafety regulations in place. age would be preventable if Golden Rice were Meanwhile introgression of selected Golden Rice adopted on a wide scale (Stein et ál., 2007; Stein lines into locally adapted varieties is very advanced et ál., 2006; Zimmermann and Ahmed, 2006; Zim- in the Philippines and in India. First fi eld trials are mermann and Qaim, 2004). The recovery on in- expected for the end of 2007. Preliminary results vestment for this intervention would be very fast from bioavailability trials indicate that β-carotene and maintenance costs minimal compared to from Golden Rice is converted into vitamin A at present interventions. even better ratios than expected. The institutions involved are working hand in hand with regulators The whole issue has an important ethical com- towards approval of Golden Rice for widespread ponent to it, as stated by the Nuffi eld Council on use. But distribution to farmers may still lie several , which concluded in their 2004 report that years away, as details of the required tests and the ‘the European Union is ignoring a moral imperative costs attached are not yet known. to promote genetically modifi ed crops for their great potential for helping the developing world’, and that In any case, it is hoped that pragmatism will they believed EU regulators had not paid enough prevail and that the costs will stay sensibly below attention to the impact of EU regulations on agricul- those of commercial crops, like Bt (Ra- ture in developing countries (Nuffi eld, 2003). maswami and Pray, 2007). 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