Molecular characterization of marker-free transgenic lines of indica rice that accumulate carotenoids in seed endosperm

A single Agrobacterium strain harbouring two binary plasmids was successfully used for the first time to develop a marker-free transgenic rice of improved nutritional value. Sixty-eight T-0 co-transformants were obtained in three indica rice cultivars-two popular high-yielding Bangladeshi varieties (BR28 and BR29), and one high-iron rice cultivar (IR68144). Marker-free lines were obtained from 14 out of 24 selected co-transformants screened in the T-1 generation. The accumulation of total carotenoids in polished T-2 rice seeds of the primary transgenic VPBR29-17-37 reached levels of up to 3.0 mu g/g, with the level of beta-carotene reaching 1.8 mu g/g. In the cultivars BR28 and IR68144, total carotenoid levels in the transformants reached 2.0 mu g/g of polished rice seeds. The levels of lutein and other carotenoids in the seeds were also significantly enhanced. T-1 plants obtained from primary transgenics with simple gene-integration patterns tended to have a lower carotenoid content than the original parental lines. This study describes the development of marker-free transgenic rice lines containing high levels of carotenoids, and addresses the relationship between the rearrangement of transgenes and the presence of metabolic end products in transgenic rice.