Marker-free transgenic (MFT) near-isogenic introgression lines (NIILs) of 'golden' indica rice (cv. IR64) with accumulation of provitamin A in the endosperm tissue

We have developed near-isogenic introgression lines (NIILs) of an elite indica rice cultivar (IR64) with the genes for β-carotene biosynthesis from dihaploid (DH) derivatives of golden japonica rice (cv. T309). A careful analysis of the DH lines indicated the integration of the genes of interest [phytoene synthase ( psy ) and phytoene desaturase ( crtI )] and the selectable marker gene (hygromycin phosphotransferase, hph ) in two unlinked loci. During subsequent crossing, progenies could be obtained carrying only the locus with psy and crtI , which was segregated independently from the locus containing the hph gene during meiotic segregation. The NIILs (BC₂F₂) showed maximum similarity with the recurrent parent cultivar IR64. Further, progenies of two NIILs were devoid of any fragments beyond the left or right border, including the chloramphenicol acetyltransferase ( cat ) antibiotic resistance gene of the transformation vector. Spectrophotometric readings showed the accumulation of up to 1.06 µg total carotenoids, including β-carotene, in 1 g of the endosperm. The accumulation of β-carotene was also evident from the clearly visible yellow colour of the polished seeds.