Executive Summary : | Micronutrients required for the normal functioning of the human body are derived from the diet, as the body cannot synthesize these micronutrients. Millets are naturally rich in micronutrients and are therefore recommended for addressing hidden hunger. However, the grain phytic acid content is a potential antinutrient that chelates the micronutrients, thereby reducing their bioavailability to the human body when consumed. Among millets, foxtail millet has the highest grain phytic acid content. In the previously funded sERB-ECRA project, the global germplasm of foxtail millet was screened to understand the variation in grain phytic acid. Also, the extremes (high and low phytic acid accessions) were identified, and expression profiling of genes involved in phytic acid biosynthesis was performed at different stages of seed development in these accessions. Based on this data, IPK1 (inositol 1,3,4,5,6-pentakis phosphate 2-kinase) gene was chosen for targeted editing using CRIsPR/Cas9 approach. Based on the expression data, IPK1 (Inositol 1,3,4,5,6-pentakis Phosphate 2-Kinase) was chosen for targetted editing using CRIsPR/Cas9 approach in the high phytic acid line (acc. G464; 2.8g/100g seed). The T0 lines were analyzed for the presence of Cas9, followed by editing in the IPK1 gene. Two independent T0 lines showed deletions in the IPK1 gene; however, both the plants are in the heterozygous condition and Cas9 positive. Phytic acid content in the grains of edited lines showed a 2- to 3-fold reduction compared to wild-type (unpublished data). This data is encouraging; however, the information is from the T0 generation and needs to be confirmed in later generations' homozygous and Cas9-free lines. Also, it is imperative to perform in vitro bioaccessibility and in vivo bioavailability assays (using mouse model), wherein the determination of micronutrient distribution in tissues post-consumption, digestion assays, and rate of excretion of phytic acid and micronutrients need to be measured. Given this, the objectives of the proposed project are framed as (i) identification of homozygous Cas9-free stable ipk1 mutants (T1 and T2 lines); (ii) characterization of ipk1 mutants for phytic acid, grain micronutrient, and other related traits, and (iii) in vitro bioaccessibility and in vivo bioavailability assays of ipk1 mutants and wild-type grains. The long-term goal of this project is to release the scientifically validated low phytic acid lines for commercial applications, following DBT's regulatory guidelines, as the mutant Cas9-free plants are now considered non-GMOs. |