Investigation of Baseline Iron Levels in Australian Chickpea and Evaluation of a Transgenic Biofortification Approach

Abstract

Iron deficiency currently affects over two billion people worldwide despite significant advances in technology and society aimed at mitigating this global health problem. Biofortification of food staples with iron (Fe) represents a sustainable approach for alleviating human Fe deficiency in developing countries, however, biofortification efforts have focused extensively on cereal staples while pulses have been largely overlooked. In this study we describe a genetic engineering (GE) approach to biofortify the pulse crop, chickpea (Cicer arietinum L.), with Fe using a combination of the chickpea nicotianamine synthase 2 (CaNAS2) and soybean (Glycine max) ferritin (GmFER) genes which function in Fe transport and storage, respectively. This study consists of three main components: (1) the establishment for baseline Fe concentration of existing germplam, (2) the isolation and study of expression pattern of the novel CaNAS2 gene, and (3) the generation of GE chickpea overexpressing the CaNAS2 and GmFER genes. Seed of six commercial chickpea cultivars was collected from four different field locations in Australia and assessed for seed Fe concentration. The results revealed little difference between the cultivars assessed, and that chickpea seed Fe was negatively affected where soil Fe bioavailability is low. The desi cultivar HatTrick was then selected for further study. From it, the CaNAS2 gene was cloned and its expression in different tissues examined. The gene was found to be expressed in multiple vegetative tissues under Fe-sufficient conditions, suggesting that it may play a housekeeping role in systemic translocation of Fe. Two GE chickpea events were then generated and the overexpression of the CaNAS2 and GmFER transgenes confirmed. Analysis of nicotianamine (NA) and Fe levels in the GE seeds revealed that NA was nearly doubled compared to the null control while Fe concentration was not changed. Increased NA content in chickpea seed is likely to translate into increased Fe bioavailability and may thus overcome the effect of the bioavailability inhibitors found in pulses; however, further study is required to confirm this. This is the first known example of GE Fe biofortified chickpea; information gleaned from this study can feed into future pulse biofortification work to help alleviate global Fe deficiency.

Opportunities for plant‐derived enhancers for iron, zinc, and calcium bioavailability: A review

“…Some soluble, low MW element‐binding ligands involved in plant mineral metabolism and transport have received some attention in bioavailability enhancement. An example is nicotianamine (NA), a nonspecific element chelator known to accumulate in high concentrations in legume seeds including chickpea (Tan et al., 2018) and soybean (Nozoye et al., 2014). NA is known for its high affinity for divalent element cations, particularly ferrous iron (Fe 2+ ) while its biosynthetic precursor, 2′‐deoxymugineic acid (DMA), can chelate Fe 3+ (Reichman & Parker, 2002; Tsednee, Huang, Chen, & Yeh, 2016).…”

Robust Genetic Transformation System to Obtain Non-chimeric Transgenic Chickpea

“…The protocol was robust enough to successfully generate stress tolerant chickpea lines with BAG genes isolated from Arabidopsis and Tripogon loliiformis , respectively. Furthermore the protocol was used to transform chickpea with multiple genes ( Nas2 , Ferritin and NPT-II placed in the same construct to generate biofortified chickpea (Tan et al, 2018).…”

“…These modifications included replacing the NOS promoter with the S1 promoter from subterranean clover stunt virus DNA segment (Schünmann et al, 2003) to drive the neomycin phosphotransferase II gene (NPTII) and inserting the CaMV35S promoter between Stu I and Sma I sites to drive genes of interest. Details of all the binary vectors used here and their transformation into Agrobacterium tumefaciens strain AGL1 were described previously (Tan et al, 2018). The genes in the different T-DNAs are shown schematically in Figure 2.…”

Biofortification of Crops Using Biotechnology to Alleviate Malnutrition

“…Other crop species have also been studied for iron and zinc biofortification using biotechnology. Tan et al [27] improved iron levels in the pulse crop chickpea (Cicer arietinum L.) by increasing iron transport and storage through a combination of chickpea nicotianamine synthase 2 (CaNAS2) and soybean (Glycine max) ferritin (GmFER) genes. Transgenic chickpea plants that overexpressed these genes illustrated a doubling of NA concentration, suggesting an increase in iron bioavailability.…”