Abstract
Phytic acid (PA), the major phosphorus reserve in soybean seeds (60–80%), is a potent ion chelator, causing deficiencies that leads to malnutrition. Several forward and reverse genetics approaches have ever since been explored to reduce its phytate levels to improve the micronutrient and phosphorous availability. Transgenic technology has met with success by suppressing the expression of the PA biosynthesis-related genes in several crops for manipulating their phytate content. In our study, we targeted the disruption of the expression of myo-inositol-3-phosphate synthase (MIPS1), the first and the rate limiting enzyme in PA biosynthesis in soybean seeds, by both antisense (AS) and RNAi approaches, using a seed specific promoter, vicilin. PCR and Southern analysis revealed stable integration of transgene in the advanced progenies. The transgenic seeds (T4) of AS (MS14-28-12-29-3-5) and RNAi (MI51-32-22-1-13-6) soybean lines showed 38.75% and 41.34% reduction in phytate levels respectively, compared to non-transgenic (NT) controls without compromised growth and seed development. The electron microscopic examination also revealed reduced globoid crystals in the Protein storage vacoules (PSVs) of mature T4 seeds compared to NT seed controls. A significant increase in the contents of Fe2+ (15.4%, 21.7%), Zn2+ (7.45%, 11.15%) and Ca2+ (10.4%, 15.35%) were observed in MS14-28-12-29-3-5 and MI51-32-22-1-13-6 transgenic lines, respectively, compared to NT implicating improved mineral bioavailability. This study signifies proof-of-concept demonstration of seed-specific PA reduction and paves the path towards low phytate soybean through pathway engineering using the new and precise editing tools.
Highlights
Soybean (Glycine max L.) is among the most nutritious and economical foods but has a relatively limited consumption and cultivation, accredited to the presence of high levels of Phytic acid (PA) accounting to ~2% of the total seed dry weight[1]
The first committed step catalyzed by MIPS involves the formation of inositol-6-phosphate from glucose 6-phosphate followed by sequential phosphorylation at the remaining five positions of the inositol ring in an ordered manner through various enzymes[23]
Cotyledonary node method was used to transform MIPS1 constructs into soybean and regenerated explants were selected on media containing different concentration of glufosinate (Fig. 4)
Summary
Soybean (Glycine max L.) is among the most nutritious and economical foods but has a relatively limited consumption and cultivation, accredited to the presence of high levels of PA (myo-inositol hexakisphosphate, IP6) accounting to ~2% of the total seed dry weight[1]. Mutations that blocked the synthesis or accumulation of PA during seed development could result in 50–95% decreased seed phytate crops with homozygous low phytate alleles (lpa) and increased Pi availability[4,10,11,12,13]. Down regulation of MIPS gene expression under CaMV35S promoter using self - complementary hairpin RNA was carried out in soybean[17] resulting in a drastic reduction (~94.5%) of phytate content in the developed transgenic lines. In this study, we attempted to generate transgenic soybean plants by silencing the MIPS1 under a seed-specific promoter, vicilin through both AS and RNAi approaches. The ultrastructure studies by transmission electron microscopy revealed a distinct reduction in the phytate globoids in these transgenics
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
