Generation and characterization of thiamin-biofortified plants

Thiamin biofortification of food crops by genetic engineering has the potential to solve human health issues related to thiamin deficiencies. Rice is an obvious target for thiamin biofortification because white rice, which is obtained after removing the thiamin-rich outer layer of the grain, is a poor source of thiamin. To increase the levels and stability of thiamin in white rice, engineering strategies that boost the expression of biosynthesis genes and/or thiamin binding proteins have been reported. In this chapter, we describe protocols for generating and characterizing thiamin-engineered plants: (1) design and building of plant expression vectors, (2) introduction of DNA constructs into plant genome by Agrobacterium-mediated transformation, (3) analysis of transgene expression by quantitative reverse-transcription polymerase chain reaction, (4) quantification of thiamin biosynthesis proteins by Western blot, and (5) quantification of thiamin by HPLC.