3.4.2. Genetic Engineering

Biofortification through genetic engineering is an alternative approach when variation in the desired traits is not available naturally in the available germplasm, a specific micronutrient does not naturally exist in crops, and/or modifications cannot be achieved by conventional breeding [136,137]. This approach was supported by the availability of fully sequenced genomes in various crops in recent years. Along with increasing the concentration of micronutrients, this approach can also be targeted simultaneously for removal of antinutrients or inclusion of promoters that can enhance the bioavailability of micronutrients [93,106,138]. This approach had not only utilized genes associated with various metabolic pathways operated in plants, but also from bacteria and other organisms [139,140]. Development of transgenic crops requires a substantial investment during the initial stage, but this could be a sustainable approach that has the potential to target large populations, especially in developing countries [96,103,141].

Several crops have been successfully modified using a transgenic approach to overcome a micronutrient deficiency. For example, enhanced accumulation (3 to 4 times) of Fe was noted in rice via expression of the iron-storage protein, ferritin [142,143]. In rice, the national levels for Fe

and Zn biofortification nutrition targets were attained under field settings in the Philippines and Colombia [144]. The genetically engineered rice (golden rice) was developed to produce β-carotene (pro-vitamin A) to fight against vitamin A deficiency [145]. Recently, transgenic multivitamin corn was produced by the simultaneous modification of three distinct metabolic pathways to increase the levels of three vitamins, i.e., β-carotene (169-fold), ascorbate (6-fold), and folate (2-fold), in the endosperm, and this could pave the way to develop nutritionally complete cereals [146]. Using metabolic engineering, the folate concentration was increased in tomato and rice [85,147]. Storozhenko et al. [148] reported more than 100-fold increase in folate concentration in rice by overexpression of *Arabidopsis thaliana* pterin and para-aminobenzoate genes, precursors of the folate biosynthesis pathway, whereas Hossain et al. [149] reported a two- to four-fold increase in *Arabidopsis* by overexpression of the gene involved in pterin biosynthesis.

To the best of our knowledge, there are no examples of biofortified pulse crops developed through a transgenic approach for Fe, Zn, Se, I, carotenoids, or folates in the available literature. However, a genetic engineering approach has been applied in pulse crops for improvement of other nutritional profile. For example, the concentration of the essential amino acid methionine was significantly increased in transgenic common bean plants (up to 23%) by expression of a methionine-rich storage albumin from the Brazil nut [150], and in the concentration of methionine in transgenic lupins (up to 94%) by expressing a sunflower seed albumin gene [151].

In recent years, targeted gene editing technologies using artificial nucleases, zinc finger nucleases (ZFNs), transcription activator-like e ffector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system (CRISPR/Cas9) have given rise to the possibility to precisely modify genes of interest, and thus have potential application for crop improvement [152,153]. These technologies have been used in various crops including rice [154,155], wheat [156], and tomatoes [157]. Recently, CRISPR/Cas9 and TALENs technologies were used to generate mutant lines for genes involved in small RNA processing of *Glycine max* and *Medicago truncatula* [158]. Similarly, CRISPR/Cas9-mediated genome editing technology was used in cowpeas to successfully disrupt symbiotic nitrogen fixation (SNF) gene activation [159]. These findings pave the way for applicability of use of gene editing technologies for various traits of interest in legumes.
