Rice is one of the major cereal crops and the cheapest source of food energy for 50% of the total world population, predominantly in the developing countries, but it is deficient in many essential micronutrients (including iron, zinc, and vitamin A). A diet based on milled rice leads to malnutrition, with deficiencies being most severe in iron, lysine, iodine, vitamin A, and zinc (FAO, 1993).

Iron deficiency is the most widespread nutritional disorder in the world. Its effects on human health are severe, and it is estimated that more than 3.5 billion people in the developing world are anemic (Ahman et al. 2000), mostly children and women.

Attempts have been made by different national, international and non-government organizations to alleviate the severity of iron-deficiency anemia by directly increasing iron intake through dietary supplementation, fortification, and food diversification. For various reasons, none of the intervention strategies has been very successful in reducing the prevalence of irondeficiency anemia in developing countries. Iron deficiency is therefore a major public health problem.

Ferritin is an iron-storage protein found in plants, animals, and bacteria, which have ferroxidase activity. Increasing the iron content in rice by introducing the ferritin gene by genetic engineering has been reported earlier in rice (Goto et al. 1999; Lucca et al.. 2001; Vasconcelos et al. 2003).

Immature embryos (10-12 days after pollination) and calli derived from mature seeds were used as transformation material. The plasmid pGPTV-bar/Fer, which contains the ferritin gene, was introduced into widely cultivated (in Bangladesh) popular rice variety BRRI Dhan 29, driven by the endosperm-specific glutelin promoter via the particle bombardment method as described previously (Vasconcelos et al. 2003). Iron content in the seeds was determined by the ICP (Inductively Coupled Argon Plasma Spectrometer).

A total of 368 putative primary transformed plants (T₀) were regenerated after bombardment and calli selection. PCR analysis was used to screen the primary transformants, and eight ferritin-positive transgenic plants (T₀) were obtained. The independent transformation event and integration pattern of the ferritin gene were confirmed by Southern blot analysis (Fig. 1). In the subsequent segregation (T₁), the ferritin gene (0.8 kb) was inherited in a typical Mendelian segregation ratio (3:1). Apart from the expected size of 0.8 kb for the gene, other larger (>0.8 kb) bands were also observed. However, the phenotypic expression of the putative transgenic plants appeared to be healthy in the transgenic greenhouse vis-a-vis the control plants. More than 95% of the transgenic plants showed normal flowering and produced fertile

seeds.

T₁ seeds obtained from T₀ plants were used to analyze the iron levels of transformed and non-transformed control plants. The iron content in the polished control seeds was 3.3 mg/kg, whereas it ranged from 4.5 to 8.9 mg/kg in the transgenic seeds (Fig. 2). Transgenic seeds of some lines showed about a two fold higher iron content than the control seeds after polishing.

This is the first report on transferring the soybean ferritin gene with an endosperm-specific promoter to an elite indica rice cultivar, BRRI Dhan 29. This finding suggests that the rice lines with enhanced iron content developed by genetic engineering may help overcome the iron-deficiency nutritional problem of the population that consumes rice as a staple food.

Acknowledgments

The work at IRRI has been generously supported by the USAID Golden Rice Project. The seed materials were obtained through international collaboration between IRRI and BRRI (Bangladesh Rice Research Institute). We thank Dr. N. Baisakh for scientific discussion and Dr. Bill Hardy for editorial assistance.

References

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FAO, 1993. Rice in human nutrition. FAO Food and Nutrition Series No. 26. FAO, Rome.

Goto, F., T. Yoshihara, N. Shigemoto, S. Toki and F. Takaiwa, 1999. Iron fortification of rice seed by the soybean ferritin gene. Nat. Biotechnol. 17: 282-286.

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Vasconcelos, M., K. Datta, N. Oliva, M. Khalekuzzaman, L. Torrizo, S. Krishnan, M. Oliveira, F. Goto and S.K. Datta, 2003. Enhanced iron and zinc accumulation in transgenic rice with the ferritin gene. Plant Sci. 164: 371-
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