Page 2: Some risk assessment data for Golden Rice
The biology of Oryza sativa (Rice)
1. Species composition and distribution
Northern India, Southeast Asia, and southern China are believed to be the centre of origin of Asian rice (Oryza sativa). The rice genus Oryza has a pantropical distribution and comprises approximately 23 species that include both diploids (2n=2x=24) and tetraploids (2n=4x=48), and ten different genome types: AA, BB, CC, BBCC, CCDD, EE, FF, GG, JJHH, and JJKK (Vaughan 1994; Ge et al 1999). The genus Oryza is distributed in Asia (eg, O. rufipogon and O. nivara, both AA genomes), Africa (eg O. barthii, and O. longistaminata, both AA genomes), Australia (eg, O. meridionalis, AA genome), tropical America (O. glumaepatula, AA genome, and O. grandiglumis, O. alta, and O. latifolia, all CCDD genomes) (Akimoto 1998; Sano and Sano 1990; Vaughan 1994). Asian cultivated rice (O. sativa) has the diploid AA genome. Wild progenitors of African cultivated rice (O. glaberrima, AA genome) are grasses endemic to West Africa.
2. Environmental safety considerations
Outcrossing and weediness potential
Cultivated rice (Oryza sativa L.) is primarily an autogamous, self-pollinating plant, although gene introgression into other cultivated rice is possible. Cultivated rice is an annual crop, it does not shatter or disperse its seed, and it has not acquired extended dormancy. Reported outcrossing rates are less than one percent and are limited by the biological characteristics of rice (Messeguer et al 2001). Factors including flower morphology, inability of pollen to remain viable longer than a few minutes, and a lack of insect vectors for pollen spread contribute to the low propensity of rice to cross-pollinate. Modern rice cultivars are often grown near older, traditional landraces in Asia, whereby only very low hybridisation rates between these two groups have been observed (Rong et al 2004). This is consistent with recommended distances of six metres, and even less in certified seed production (Gealy et al 2003). Oryza species with different genome types have significant reproductive isolation, making them unlikely to hybridise with each other. Hybridisation between species in different genera within the tribe Oryzeae is extremely difficult, even using artificial conditions, such as embryo rescue.
In the United States, the only wild species known to be compatible with cultivated rice is O. rufipogon, which has been found in a single location in the Everglades of Florida, and red rice, a wild variant of cultivated O. sativa, thus it is considered very unlikely that cultivated rice would hybridise with O. rufipogon under such conditions.
Red rice, also known as O. sativa f. spontanea, is considered a weedy species in the cultivation of rice, as the reproduction of red rice favours specific environmental conditions (such as flooded fields) that are typical in the cultivation of commercial rice. Outside of rice production areas, red rice is not a weed species. Gene flow from cultivated rice into red rice can occur, although the rate is likely to be very low with levels being dependent on the degree of overlapping of flowering periods. Weedy rice is readily found in tropical America. Weedy rice appears to be mainly composed of annual Oryza spp with feral traits including seed shattering. In contrast to Asia, where manual transplanting is still predominant, direct seeding of weedy rice-contaminated seed is common for a high proportion of rice farmers in tropical America, ensuring field reinfestations and making it one of the most serious weed problems in this region (Fischer and Ramirez 1993).
Weedy rice is often referred to as red rice because of the red color of its pericarp, and it has been botanically classified as O. sativa f. spontanea, the same species as cultivated rice (Chu et al 1969; Diarra et al 1985; Ellstrand et al 1999; Langevin et al 1990; Oka and Chang 1961). Reports suggest that weedy rice may include other Oryza species, including O. barthii, O. glaberrima, O. longistaminata, O. nivara, O. punctata, O. sativa, and O. latifolia (an American tetraploid) (Holm et al 1997). Hybrid swarms between the American form of O. perennis and O. sativa have been found in Cuba (Chu and Oka 1969). Weedy rice may also have evolved through the dedomestication of cultivated rice to weedy types (Vaughan et al 2003). In addition to seed shattering, weedy rice seeds may possess secondary dormancy, and some types are morphologically indistinguishable from rice varieties yet still shatter seed (Lentini and Espinoza 2005). Natural gene flow estimates in the field from herbicide-resistant rice into weedy rice under temperate conditions indicate hybridisation rates of under one percent (Chen et al 2004; Estorninos et al 2002; Messeguer et 2004; Zhang et al 2003), as confirmed by genetic analysis. However, a cumulative hybridisation rate(over a 3-year period) under temperate conditions may be from 1 to 52% (Guadaggnuolo et al 2001), indicating that genes from rice varieties may transfer and be quickly fixed into weedy rice if they have a selective value. The cumulative rate of introgression may be even higher under tropical conditions, because of the lack of crop rotation and several crop cycles per year. Several biological, genetic, and environmental factors affect the level of outcrossing compatibility, including temperature, humidity, genotype, flower morphology, stigma receptivity, pollen viability, pollen germination, and pollen tube development.
3. Food and Feed Safety Considerations
Antinutrients in rice
Rice contains a small number of antinutritional factors that are concentrated in the bran fraction and which, except for phytic acid, are subject to heat denaturation (inactivation). These antinutrients include: phytic acid, which is a storage form of phosphorus in plant seeds that also chelates calcium, zinc, iron, and magnesium in the digestive tract of animals, thus interfering with the absorption of these nutrients; trypsin inhibitor; and lectins, which are a class of proteins with specific binding affinities for particular carbohydrate moieties of glycoproteins present in cell walls and cell plasma membranes, and which have been associated with a range of antinutritive effects and some disease pathologies.
Phytin is an organic phosphorus compound contained primarily in the bran layer, and it exists as a mixture of calcium-magnesium salts of phytic acid. Free phytic acid (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) chelates nutritional metal ions such as calcium and iron, which reduces the absorbability of these ions into the body (Thompson and Weber 1981). It has been reported that phytic acid reduced platelet aggregation and had an inhibitory effect against blood clot formation which may cause thrombosis and atherosclerosis (Vucenik et al 1999). Phytic acid is considered to be an anticarcinogen influencing signal transduction pathways, cell cycle regulatory genes, differentiation genes or suppressor genes (Shamsuddin 1999).
Oryzacystatin has been isolated from rice bran (Abe et al 1987) and is considered a cysteinyl proteinase inhibitor (cystatin). It is inactivated by heat above 120°C (Juliano 1993).
Lectins are carbohydrate-binding proteins which agglutinate cells that are able to precipitate glycoconjugates or polysaccharides (Goldstein et al 1980). The toxicity of lectins is due to their ability to bind to specific carbohydrate receptor sites on the intestinal mucosal cells and interfere with the absorption of nutrients across the intestinal wall (Liener 1986). Rice bran lectin, haemagglutinin, has been found to be associated with agglutination of human A, B and O group receptors with specific binding to 2-acetamido-2-deoxy-D-glucose (Poola 1989). Rice bran lectin is heat labile at temperatures above 80°C (Ory et al 1981; Poola 1989). Mannnose-binding rice lectin is distributed in all parts of the rice plant, and it has a potential ability to agglutinate bacterial cells of Xanthomonas campestris pv oryzae, the pathogen causing bacterial leaf blight in rice, and also spores and protoplasts of Magnaporthe grisea, the rice blast fungus (Hirano et al 2000).
While rice is not considered to be a common cause of allergic reactions to food, allergic reactions have been documented, and certain proteins in rice have been identified as allergens. The first reported allergens in rice were 14-16 kDa proteins which were detected using sera from patients allergic to rice (Matsuda et al 1991). A 16 kDa protein was later recognised as a major rice allergen. This protein has significant amino acid homology to barley trypsin inhibitor and wheat alpha-amylase inhibitor (Izumi et al 1992). Subsequently, rice seed proteins with molecular masses of 26, 33, and 56 kDa have been recognised as being allergenic. The 33 kDa protein has been recently characterised and identified as the enzyme glyoxalase I (Usui et al 2001).
A trypsin inhibitor has been isolated from rice bran and characterised (Tashiro and Maki 1979). There seems to be no standard way of reporting the quantity of the inhibitor, and it does appear to be heat labile. No trypsin inhibitor was detected in the grain or polished rice, but in the bran (AgrEvo 1999).
Alpha-amylase Subtilisin Inhibitor:
The amino acid sequence of the bifunctional alpha-amylase subtilisin inhibitor from rice is known (Ohtsubo and Richardson 1992). Bifunctional inhibitors have been proposed to be associated with defence of the seed against insect pests and pathogenic microorganisms (Ryan 1990).
- Abe K, Emori Y, Kondo H, Suzuki K, Arai S (1987) Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). J Biol Chem 102:16793-16797.
- AgrEvo. 1999. Safety, compositional and nutritional aspects of LibertyLink rice transformation events LLRICE06 and LLRICE62. US FDA/CFSAN BNF 63.
- Akimoto M (1998) Population genetic structure of wild Oryza glumaepatula distributed in the Amazon flood area influenced by life history traits. Mol Ecol 7:1371-1381.
- Chen LJ, Lee DS, Song ZP, Suh HS, Lu BR (2004) Gene flow from cultivated rice (Oryza sativa) to its weedy and wild relatives. Ann Bot 93:67-73.
- Chu YE, Morishima H, Oka HI (1969) Reproductive barriers distributed in cultivated rice species and their wild relatives. Jpn J Genet 44:207-223.
- Chu YE, Oka HI (1969) Introgression across isolating barriers in wild and cultivated Oryza species. Evolution 24:344-355.
- Diarra A, Smith R, Talbert R (1985) Growth and morphological characterization of red rice (Oryza sativa L.) biotypes. Weed Sci 33:310-314.
- Ellstrand NC, Prentice HC, Hancock JF (1999) Gene flow and introgression from domesticated plants into their wild relatives. Ann Rev Ecol Syst 30:539-563.
- Estorninos LE Jr, Gealy DR, Dillon TL, Baldwin EL, Burgos RR, Tai TH (2002) Determination of hybridization between rice and red rice suing microsatellite markers. Proc South Weed Sci Soc 55:197-198.
- Fischer AJ, Ramírez A (1993) Red rice (Oryza sativa): competition studies for management decisions. Int J Pest Mgmt 39:133-138.
- Ge S, Sang T, Lu BR, Hong DY (1999) Phylogeny of rice genomes with emphasis on origins of allotetraploid species. Proc Natl Acad Sci USA 96:14400-14405.
- Gealy DR, Mitten DH, Rutger JN (2003) Gene flow between red rice (Oryza sativa) and herbicide-resistant rice (O. sativa): implications for weed management. Weed Technol 17:627-645.
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- Guadaggnuolo R, Savova-Bianchi D, Felber F (2001) Gene flow from wheat to jointed goat grass, as revealed by RAPD and microsatellite markers. Theor Appl Genet 103:1-8.
- Hirano K, Teraoka T, Yamanaka H, Harashima A, Kunisaki A, Takahashi H, Hosokawa D (2000) Novel mannose-binding rice lectin composed of some isolectins and its relation to a stress-inducible salT gene. Plant Cell Physiol 41:258-267.
- Holm L, Doll J, Holm E, Pancho J, Herbeger J (1997) The wild rices. In, World Weeds: Natural History and Distribution. New York: John Wiley & Sons pp 531-547.
- Izumi H, Adachi T, Fujii N, Matsuda T, Nakamura R, Tanaka K, Urisu A, Kurosawa Y (1992) Nucleotide sequence of a cDNA clone encoding a major allergenic protein in rice seeds. Homology of the deduced amino acid sequence with members of alpha-amylase/trypsin inhibitor family. FEBS Lett 302: 213-216.
- Juliano BO (1993) Rice in Human Nutrition. FAO, Rome
- Langevin S, Clay K, Grace J (1990) The incidence and effects of hybridization between cultivated rice and its related weed red rice (Oryza sativa L.) Evolution 44:100-108.
- Lentini Z, Espinoza AM (2005) Coexistence of Weedy Rice and Rice in Tropical America - Gene Flow and Genetic Diversity. In, Crop Ferality and Volunteerism: A Threat to Food Security in the Transgenic Era (J Gressel, ed). CRC Press pp 303-319.
- Liener IE (1986) In, The Lectins: Properties, Functions and Applications in Biology and Medicine (IE Liener, N Sharon, IJ Goldstein, eds) Academic Press, New York, pp. 527-552.
- Matsuda T, Nomura R, Sugiyama M, Nakamura R (1991) Immunochemical studies on rice allergenic proteins. Agric Biol Chem 55:509-513.
- Messeguer J, Fogher C, Guiderdoni E, Marfa V, Catala MM, Baldi G, Mele E (2001) Field assessment of gene flow from transgenic to cultivated rices(Oryza sativa L.) using a herbicide resistance genes as tracer marker. Theor Appl Genet 103:1151-1159.
- Messeguer J, Marfa V, Catala MM, Guiderdoni E, Mele E (2004) A field study of pollen-mediated gene flow from Mediterranean GM rice to conventional rice and the red rice weed. Mol Breed 13:103-112.
- Oka HI, Chang W (1961) Hybrid swarms between wild and cultivated rice species, Oryza perennis and O. sativa. Evolution 15:418-430.
- Ory RL, Bog-Hansen TC, Mod RR (1981) Properties of hemagglutinins in rice and other cereal grains. In, Antinutrients and Natural Toxicants in Foods (RL Ory, ed). Westport, CT, USA, Food & Nutrition Press.
- Poola I (1989) Rice lectin: physicochemical and carbohydrate-binding properties. Carbohydr Polym 10: 81-288.
- Rong J, Xia H, Zhu YY, Wang YY, Lu BR (2004) Asymmetric gene flow between traditional and hybrid rice varieties (Oryza sativa) estimated by nuclear SSRs and its implication in germplasm conservation. New Phytologist 163:439-445.
- Sano Y, Sano R (1990) Variation in the intergenic spacer region of ribosomal DNA in cultivated and wild rice species. Genome 33:209-218.
- Shamsuddin AM (1999) Metabolism and cellular functions of IP6: A Review. Anticancer Res. 19(5A):3733-3736.
- Thompson SA, Weber CW (1981) Effect of dietary fiber sources on tissue mineral levels in chicks. Poultry Sci 60:840-845.
- Usui Y, Nakase M, Hotta H, Urisu A, Aoki N, Kitajima K, Matsuda T (2001) A 33-kDa allergen from rice (Oryza sativa L. japoniJn, and identification as a novel glyoxalase 1. J Biol Chem 276:11376-11381.
- Vaughan DA (1994) The Wild Relatives of Rrice. A Genetic Resources Handbook. Los Baños, Philippines: International Rice Research Institute, 137 pp.
- Vaughan DA, Morishima H, Kadowaki K (2003) Diversity in the genus Oryza. Curr Opinion Plant Biol 6:139-146.
- Vucenik I, Podczasy JJ, Shamsuddin AM (1999) Antiplatelet activity of inositol hexaphosphate (IP6). Anticancer Res 19(5A):3689-3693.
- Zhang N, Linscombe S, Oard J (2003) Out-crossing frequency and genetic analysis of hybrids between transgenic glufosinate herbicide-resistant rice and the weed, red rice. Euphytica 130:35-45.
The only difference between Golden Rice and other rice varieties is that it is able to produce caroteonoids not only in the leaves but also in the grains.
Page 1: Basics of biosafety assessment
Page 2: Some risk assessment data for Golden Rice