CRISPR/Cas9-mediated disruption of the rice OsVIT1 and OsVIT2 genes to improve grain iron density

O'Brien M1, Kielnhofer EOF1, Eftekhari FN2 and Johnson AAT1

  1. School of Biosciences, The University of Melbourne, VIC 3010, Australia.
  2. Centre for Tropical Crops and Biocommodities, Queensland University of Technology, QLD 4001, Australia.

Rice provides 3.5 billion humans with more than 20% of their daily caloric intake. The commonly consumed white rice of modern rice cultivars is inherently poor in iron (Fe). Human Fe deficiency can cause mild to severe anaemia and is the most common nutritional disorder worldwide, estimated to affect up to two billion people. It is most common in developing countries where people depend upon micronutrient-poor staple crops such as rice. This has prompted the development of biofortified rice varieties with enhanced grain Fe density as a low-cost and sustainable strategy to tackle global human Fe deficiency. The rice VACUOLAR IRON TRANSPORTER 1 and 2 genes (OsVIT1/OsVIT2) are ubiquitously expressed at low levels in all tissues except the flag leaf where they are predominantly expressed. The OsVIT1 and 2 proteins are Fe transporters that are localised to the vacuole where they function to sequester Fe in the flag leaf. Disruption of OsVIT1 or OsVIT2 gene through T-DNA insertion lead to an increased in Fe concentrations in all tissues, including the grain1. In this study, we have used Agrobacterium-mediated transformation of rice with CRISPR/Cas9 plasmids targeting the OsVIT1 and OsVIT2 genes to generate a collection of osvit1 and osvit2 mutants. Eleven and fifteen independent mutant alleles were generated for OsVIT1 and OsVIT2, respectively. We present molecular and phenotypic analyses of homozygous osvit1 and osvit2 mutants, including an analysis of grain Fe concentration. 1 Zhang et al, 2012. Plant Journal 72, 400-410.