Rice callus as a high-throughput platform for synthetic biology and metabolic engineering of carotenoids.

Carotenoids are a large class of important lipid-soluble phytonutrients that are widely used as nutritional supplements due to their health-promoting activities. For example, β-carotene is the precursor for vitamin A synthesis, and astaxanthin is a powerful antioxidant. However, these carotenoids cannot be synthesized de novo by humans. These properties of β-carotene and astaxanthin make them attractive targets for metabolic engineering in rice (Oryza sativa) endosperm because rice is an important staple food in developing countries, and rice endosperm is devoid of carotenoids. In this chapter, we introduce an assay based on rice embryogenic callus for the rapid functional characterization of genes involved in carotenoid biosynthesis and accumulation. The system is also an ideal platform to characterize cereal endosperm specific promoters. Four diverse cereal endosperm specific promoters were demonstrated to be active in rice callus despite their restricted activity in mature plants. The use of endosperm specific promoters that are expressed in rice callus, but remain silent in regenerated vegetative tissue, directs accumulation of carotenoids in the endosperm without interfering with plant growth. Rice callus is a useful platform for improving gene editing methods and for further optimizing pathway engineering. Thus, the rice callus platform provides a unique opportunity to test strategies for metabolic engineering of synthetic carotenoid pathways, leading to novel carotenoid-biofortified crops.