No abstract

Chickpea transformation is an important component for the genetic improvement of this crop, achieved through modern biotechnological approaches. However, recalcitrant tissue cultures and occasional chimerism, encountered during transformation, hinder the efficient generation of transgenic chickpeas. Two key parameters, namely micro-injury and light emitting diode (LED)-based lighting were used to increase transformation efficiency. Early PCR confirmation of positive in vitro transgenic shoots, together with efficient grafting and an extended acclimatization procedure contributed to the rapid generation of transgenic plants. High intensity LED light facilitate chickpea plants to complete their life cycle within 9 weeks thus enabling up to two generations of stable transgenic chickpea lines within 8 months. The method was validated with several genes from different sources, either as single or multi-gene cassettes. Stable transgenic chickpea lines containing GUS ( uidA ), stress tolerance ( AtBAG4 and TlBAG ), as well as Fe-biofortification ( OsNAS2 and CaNAS2 ) genes have successfully been produced.

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.

Iron deficiency is a major problem in both developing and developed countries, and much of this can be attributed to insufficient dietary intake. Over the past decades several measures, such as supplementation and food fortification, have helped to alleviate this problem. However, their associated costs limit their accessibility and effectiveness, particularly amongst the financially constrained. A more affordable and sustainable option that can be implemented alongside existing measures is biofortification. To date, much work has been invested into staples like cereals and root crops—this has culminated in the successful generation of high iron-accumulating lines in rice and pearl millet. More recently, pulses have gained attention as targets for biofortification. Being secondary staples rich in protein, they are a nutritional complement to the traditional starchy staples. Despite the relative youth of this interest, considerable advances have already been made concerning the biofortification of pulses. Several studies have been conducted in bean, chickpea, lentil, and pea to assess existing germplasm for high iron-accumulating traits. However, little is known about the molecular workings behind these traits, particularly in a leguminous context, and biofortification via genetic modification (GM) remains to be attempted. This review examines the current state of the iron biofortification in pulses, particularly chickpea. The challenges concerning biofortification in pulses are also discussed. Specifically, the potential application of transgenic technology is explored, with focus on the genes that have been successfully used in biofortification efforts in rice.