Interference for MIPS1 mRNA results in biofortified lablab (Lablab purpureus L.) lines having reduced phytic acid levels

Anti-nutritional factor phytic acid, which causes reduced mineral bioavailability by forming indigestible complexed minerals and charged proteins in livestocks and humans, is a major dietary constraint. Gene Myo-inositol 1-phosphate synthase (MIPS) codes for the enzyme L-myo-inositol 1-phosphate synthase, which isomerizes D-glucose-6-phosphate to D-inositol 3-phosphate, the precursor for phytate (myo-inositol hexakisphosphate, InsP₆). Lablab pods at the harvestable stage contain a high level of phytic acid. This work describes the development of lablab lines with low phytic acid content through RNA interference of MIPS1 gene. Sense- and antisense fragments of the target region in the MIPS1 gene were PCR amplified using specific primers and treated with T4 DNA polymerase and dATP. The pRNAi-LIC vector was digested with Sma 1, and treated with T4 DNA polymerase and dTTP. The sense- and anti-sense fragments were inserted to vector, cloned in E. coli and cloning was confirmed through PCR and restriction digestion of plasmids isolated from positive colonies. Through sequencing, the direction of target gene fragments in the vector was confirmed. The recombinant plasmid was transformed into Agrobacterium tumefaciens, and through in planta transformation, six cisgenic lines were obtained. Synthesis of target siRNA was confirmed using stem-loop Reverse Transcription PCR. Comparative expression levels of MIPS1 in RNAi lines were downregulated to 0.03–0.75-fold. The phytic acid level in mature pods was reduced significantly from 410.88 mg/100 g in wild type to 56.47–278.53 mg/100 g (32.22–86.25 % reduction). Germination, development and agronomic performance of the RNAi lines were comparable with those of wild type.