**5. Status of Biofortification**

In recent years, several crops with increased micronutrient concentration have been introduced in several developing countries, and this has helped in overcoming nutrient deficiency in the target population. For example, the introduction of the orange sweet potato biofortified with β-carotene increased vitamin A intake among children and women in Mozambique [219] and Uganda [220], and maize biofortified with provitamin A increased the concentration of vitamin A in 5–7-year-old children in Zambia who consumed it for three months [221]. Similarly, serum ferritin and total body iron were improved in iron-deficient adolescent boys and girls from Maharashtra, India, who consumed Fe-biofortified pearl millet flat bread for four months [222]. Regarding pulse crops, the consumption of Fe biofortified beans for 4.5 months improved the hemoglobin and total body iron in iron-depleted university women in Rwanda [223].

By the end of 2016, more than 150 biofortified varieties of 10 crops had been released in 30 countries, and these are consumed by more than 20 million people in developing countries [163]. To date, HarvestPlus has released or tested more than 290 varieties of 12 staple food crops including vitamin A orange sweet potato, iron beans, iron pearl millet, vitamin A yellow cassava, vitamin A orange maize, zinc rice, and zinc wheat in 60 countries (www.harvestplus.org). Iron beans are delivered in Rwanda and Democratic Republic of Congo, zinc rice is in Bangladesh, and zinc wheat is in India and Pakistan.

Among pulse crops, HarvestPlus has released 10 Fe-biofortified bean varieties each in Rwanda (RWR 2245, RWR 2154, MAC 42, MAC 44, CAB 2, RWV 1129, RWV 3006, RWV 3316, RWV 3317, and RWV 2887) and the Democratic Republic of Congo (COD MLB 001, COD MLB 032, HM 21-7, RWR 2245, PVA 1438, COD MLV 059, VCB 81013, Nain de Kyondo, Cuarentino, and Namulenga) (www.harvestplus.org). Similarly, several varieties of lentils with high iron and zinc have been released by HarvestPlus and The International Center for Agricultural Research in the Dry Areas (ICARDA) in various countries: seven in Nepal (ILL 7723, Khajurah-1, Khajurah-2, Shital, Sisir Shekhar, Simal), five in Bangladesh (Barimasur-4, Barimasur-5, Barimasur-6, Barimasur-7, and Barimasur-8), two each in India (L4704, Pusa Vaibhav) and Syria (Idlib-2, Idlib-3), and one in Ethiopia (Alemaya). For the effective delivery and production of these crops, HarvestPlus works closely with various public and private organizations [163]. For example, in Rwanda, HarvestPlus with the help of Rwanda Agriculture Board (RAB) facilitated the production of bean seeds through contracted farmers and cooperatives and acquired about 80% of certified seeds during 2011–2015.

Biofortification to enrich nutrient profile of pulse crops is one of the major goals in the pulse crop breeding program at the Crop Development Centre (CDC), University of Saskatchewan, which was established in 1971 with the objectives to improve existing crops and develop new crops (https://agbio. usask.ca/research/centres-and-facilities/crop-development-centre.php#MoreAbouttheCDC). In recent years, several projects have been undertaken to evaluate pulse crops for the profiling of folates, carotenoids, polyphenols, Fe, Zn, and Se.

### **6. Challenges and Future Strategies for Biofortification**

A greater micronutrient density and a high yield are prerequisites for e ffective biofortification, and these crops must be adopted by farmers and consumed by the target population [21]. Bouis and Saltzman [163] outlined three important challenges for HarvestPlus to reach one billion people by 2030, i.e., building consumer demand, mainstreaming biofortified traits into public and private breeding programs, and integrating biofortification into public and private policies.

Various factors such as genetic diversity in the gene pool, the reduction of antinutrients (especially phytate and polyphenols), and increasing the concentration of promoter substances including certain amino acids (cysteine, lysine, and methionine) and ascorbic acid (vitamin C), which enhance the absorption of essential minerals, and/or high yield, are key for the success of biofortification strategies [93,167].

Narrow genetic variation in the plant gene pool, a long-development time for generating cultivars with a desired trait, and the dependence on the phytoavailability of the mineral nutrients in the soil are limitations for conventional breeding approach [138].

The issue of narrow genetic variation for micronutrient concentration might be overcome by the use of wild germplasm and land races, which may contain a high variation in micronutrient concentration [93,106,138].

For e fficient biofortification, the focus should be on increasing the bioavailability of micronutrients simultaneously with increase in their concentration. This can be achieved by increasing the concentration of promoters that stimulate the absorption of minerals and by reducing the concentrations of antinutrients that interfere with absorption [93].

Vitamin E, vitamin D, vitamin C, choline, niacine, and provitamin A are considered promoter substances and stimulate the absorption of Se, Ca P, Fe, Zn, methionine, and tryptophan [224]. In contrast, certain antinutrients including phytate and certain polyphenols reduce the bioavailability of micronutrients in crops [93]. Phytate, a form of phosphorus stored in seed, is not digested by humans or monogastric animals [225]. During digestion, it can bind to iron and zinc and thus restrict their absorption [226]. The concentration of phytate can be controlled by identifying low phytate lines by germplasm screening [227], manipulating the biosynthesis of phytate via mutation of a myo-inositol kinase (MIK) gene [228], and overexpressing phytase, a phytate degrading enzyme [229].

In the recent past, low-phytate lines in pulse crops have been developed and characterized to reduce the concentration of phytate and thus improve mineral absorption [225,230–233]. Warkentin et al. [225] developed low-phytate pea lines, 1-150-81 and 1-2347-144, using chemical mutagenesis of cultivar CDC Bronco, a high-performing pea variety. They observed an approximately 60% reduction in phytate phosphorus in low-phytate lines with an increase in inorganic phosphorus. However, these lines had a slightly lower seed weight and a lower yield compared to CDC Bronco. Nevertheless, these lines are being used to breed for improvement of phosphorus and micronutrient bioavailability, along with high grain yield. Subsequently, Liu et al. [226] evaluated the effects of phytate and seed coat polyphenols on the bioavailability of iron using low-phytate pea lines (1-150-81 and 1-2347-144). The iron bioavailability (FEBIO) was 1.4–1.9 times greater in low-phytate lines compared to normal phytate varieties. Further, pigmented seed coat pea showed a seven times lower FEBIO than non-pigmented seed coats; however, the removal of seeds coats increased the FEBIO up to six times. To understand the genetic basis of the low phytic acid (*lpa*) mutation in the pea, Shunmugam et al. [232] amplified a 1530 bp open reading frame of *myo*-inositol phosphate synthase (MIPS), the rate-limiting step in the phytic acid biosynthesis pathway, from CDC Bronco and two *lpa* pea genotypes, 1-150-81 and 1-2347-144. They did not observe any difference in coding sequence in *MIPS* between CDC Bronco and *lpa* genotypes and noticed that mutation in *MIPS* did not cause the *lpa* trait in pea lines.

Various studies in common beans suggested that *lpa* lines can improve iron bioavailability by reducing the phytic acid level up to 90% [230,234]. Homozygous *lpa* mutant line (*lpa-280-10*) was isolated in common beans from a mutagenized population, and this mutant had 90% less phytic acid and higher free Fe in the seeds compared to the wild type [230]. Further, at the molecular level, it was observed that a recessive mutation was responsible for the *lpa* character. Panzeri et al. [231] mapped the *lpa1*(280-10) mutation and identified and sequenced a candidate gene in common beans for comparison with the soybean genome. They observed that the *lpa1*(280-10) mutation co-segregated with the mutated multidrug resistance-associated protein (MRP) type ATP-binding cassette transporter gene *(Pvmrp1)*, which is orthologous to the *lpa* genes of *Arabidopsis AtMRP5* and maize *ZmMRP4*. They further observed that a defective *Mrp1* gene caused an *lpa1* mutation in common beans that downregulates the phytic acid pathway at the transcriptional level and thus reduced seed myo-inositol. Recently, a new *lpa* line influencing the *PvMRP1* phytic acid transporter was identified in common beans using ethyl methane sulfonate mutagenesis [233]. Further, *PvMRP* promoters were characterized in *Arabidopsis thaliana* and *Medicago truncatula* transgenic plants.

Polyphenols are secondary metabolites including flavonoids and proanthocyanidins [235] and provide protection against various fungal pathogens [236]. They are natural sources of antioxidants in the human diet and are present in fruits, vegetables, cereals, and legumes [237,238]. Previously, all polyphenols were considered as inhibitors of Fe bioavailability in humans. A recent study by Hart et al. [239] reported that four polyphenols inhibited Fe uptake, whereas four other polyphenols promoted Fe uptake upon evaluating the effect of polyphenols present in black bean seed coats on Fe uptake using Caco-2 cells (human cell line). They further concluded that specific polyphenols (promoter of Fe uptake) can be targeted in future breeding for improved Fe bioavailability. Jha et al. [240] detected 30 polyphenols in a recombinant inbred line population developed from crossing pea cultivars CDC Amarillo (white flower) and CDC Dakota (purple flower). Among 30 polyphenols, catechin, 3,4-dihydroxybenzoic acid, and kaempferol 3-glucoside were present in all pea lines, and these were considered promoters of Fe uptake by Hart et al. [239]. Thus, promising accessions having Fe promoter polyphenols can be identified via a wide exploration of germplasms for developing cultivars with additional health benefits.

Postharvest processing can also play an important role in efficient utilization of biofortified crops, as a substantial amount of minerals from the diet can be lost by milling or polishing [241] and cooking. Therefore, efforts should be made to retain the micronutrient concentration in edible seeds, and their absorption by the consumer after processing and cooking [242]. Retention of zinc content after cooking in biofortified rice varieties produced either through traditional breeding or genetic engineering approaches has been discussed in detail by Tsakirpaloglou et al. [243].

Iodization of salt was not enough to overcome I deficiency due to several factors such as the unavailability of iodized salt for all households, the volatilization of I during cooking, and insu fficient consumption due to health issues [55,93,200,244]. Hence, for successful biofortification, further research was needed to identify traits that control uptake, mobilization, and retention of I in the plant, and these can be manipulated in plant breeding or using a genetic engineering approach [56].

In pulse crops, growth and productivity are a ffected by various abiotic and biotic stresses, which can result in significant reduction of grain yield [245–250]. These stresses can significantly alter the nutritional profile of the harvested seeds. As mentioned previously, the targeted micronutrients are either antioxidants or part of enzymes involved in various metabolic processes including electron transfer and oxidation reductions; thus, they protect cells from oxidative damage by quenching reactive oxygen species generated under environmental stresses [32,33,40,41,46,68,69]. Biofortified crops with a greater concentration of micronutrients can better withstand adverse environmental conditions and demonstrate improved adaptation under these conditions.
