Molecular protocol to develop β-carotene-biofortified rice events via molecular optimization

Providing food with nutrition and functionality is crucial for sustaining human life. Rice (Oryza sativa L.) is a representative staple crop with high carbohydrate content but low amounts of essential amino acids, micronutrients, and carotenoids such as provitamin A. To improve the nutritional quality, rice endosperm was biofortified to accumulate carotenoids such as β-carotene through genetic engineering (i.e., using synthetic carotenoid biosynthetic genes, a nonmammalian viral polycistronic sequence, and an optimized promoter and transit peptide) and high-throughput rice transformation (approximately 300 transgenic plants per construct). To facilitate the safety assessment of genetically modified food, molecular characterization was performed to select elite lines equipped with a single intergenic insertion of T-DNA, high transgene expression, in this case leading to high carotenoid content, and with phenotypic and compositional substantial equivalence. In this study, we present β-carotene-biofortified rice event candidate lines eligible for commercial use and a disclosed molecular protocol for the development of biotech rice crops.