Transgenic Techniques for Iron, Zinc, and Iodine (IZI) Bio fortification in Crops

Keywords

Introduction

Micronutrient malnutrition will become more prevalent as the world's population grows, and it currently affects over three billion people globally [1]. In persons living in low-income countries, malnutrition deficiency, sometimes known as "hidden hunger," increases the risk of infectious illness and death from diarrhoea, measles, malaria, and pneumonia (WHO, 2000) [2]. Malnutrition has severe and long-lasting repercussions, which can even be passed down from generation to generation [3]. Malnutrition during pregnancy raises the chance of death and stunts growth, resulting in Low Birth Weight (LBW) and jeopardizing the child's life [4]. Malnutrition continues to be a serious public health issue in developing and impoverished countries [5]. People's diets are less diverse now than they were 30 years ago, resulting in micronutrient deficiencies, particularly iron (Fe), zinc (Zn), and iodine (I) [6]. Fe and Zn are critical trace elements for a range of metabolic functions [7]. Deficiencies in zinc and iron were ranked fifth and sixth, respectively, among the top ten risk factors for disease burden over the world [8]. Because of blood losses during menstruation and childbirth, micronutrient deficiencies are common in children and even more so in women [9]. Furthermore, in underdeveloped nations, micronutrient insufficiency is exaggerated by a lack of understanding and affordability of various and balanced meals, dietary patterns, and a high frequency of infectious diseases [10]. Various strategies such as food diversity, pharmacological supplementation, and fortification have been emphasized to fight nutritional deficit, particularly mineral inadequacy [11].

Current malnutrition-prevention strategies

The greatest strategy to prevent and even eliminate micronutrient deficiency is to eat a well-balanced diet rich in micronutrients [12]. Supplementation, dietary fortification, homestead food production, and crop bio fortification are all conventional but effective ways to manage malnutrition and micronutrient deficiencies [13]. The most practical technique of preventing vitamin deficit in people is to fortify diet with micronutrients [14]. Food fortification, as an approach for filling dietary gaps, has the added benefit of being able to give nutrients to vast portions of the population without necessitating drastic changes in eating habits [15]. Some countries have already begun to implement vitamin A fortification of butter, margarine, and sugar, as well as iodine fortification of salt, vitamin fortified milk, and vitamin B fortified cereals, all of which have a lengthy history [16]. Food fortification that is mandated rather than optional is more effective and has been used successfully for decades around the world [17]. Fortification of milk and oil is required in 14 and 27 nations, respectively. In 134 nations, salt fortification with iodine and fluoride is also required [18]. Around 83 countries are now using fortification legislation to supplement basic foods with micronutrients [19]. However, there are certain disadvantages to current food fortification procedures [20]. Food fortification is usually only possible in nations with well-developed, well-monitored, and well-regulated pharmaceutical and food processing industries [21]. Another significant disadvantage of supplementing and fortification systems is that they have recurring expenditures year after year, and their performance is contingent on funding [22]. This strategy works especially well in countries where the poorest people supplement their diets with small amounts of processed goods [23]. Unfortunately, consumption of industrially processed foods is small in the poorest developing countries, where the majority of the poor, particularly the farming population, rely on their own products for nourishment [24]. As a result, the reach of these food fortification programmers’ may be limited, particularly in developing countries' rural areas, where the majority of the poor live [25]. In India, obligatory fortification began in 1953 with the fortification of hydrogenated vegetable oil with vitamins A and D. To manage Goiter, salt fortification with iodization was used in 1998 [26]. West Bengal was the first to fortify wheat flour in 2000, followed by the Andaman and Nicobar Islands [27]. Micronutrient deficits are treated in industrialized nations by supplementation and fortification [28]. In poorer nations, however, extensive implementation of dietary diversification and food fortification/supplementation programmes is hampered by low income and poor market access for the target population [29]. Unfortunately, none of these measures based on socioeconomic development are effective in combating micronutrient deficiency [30]. Furthermore, the success of the aforementioned tactics necessitates societal behavioral changes that rely on literacy, communication, social marketing, and repeated investments [31]. Numerous investigators' global experience has confirmed that, rather than a single more expensive technique, a combination of several less expensive approaches is required to prevent nutritional shortage [32]. Plant scientists are concentrating their efforts to develop methods to apply fertilizers and/or use plant breeding strategies to increase the concentrations and/or bioavailability of mineral elements in the edible portion of crop plants to address the occurrence of micronutrient deficiencies in human populations [33]. Bio fortification methods are classified as 'agronomic' (fertilizer-based) and 'genetic' (breeding-based) [34]. Bio fortification is a method of increasing the micronutrient content of agricultural produce by targeting and modulating mineral nutrient movement pathways (root uptake, transport, remobilization, storage, and enhanced bioavailability), 'pulling' nutrients from the soil and 'pushing' them to economically important parts of the plant in bioavailable forms [35]. Agronomic bio fortification, traditional plant breeding, and genetic engineering are the three basic methodologies for bio fortifying food crops that have been used to date [36]. The main focus of agronomical methods is on optimizing mineral fertilizer application and/or improving mineral element solubilization and mobilization in the soil [37]. By lowering 'anti-nutrient' concentrations, the latter two techniques aim to generate and/or improve plant cultivars with greater micronutrient accumulation capability and increased levels of bioavailability [38]. To overcome the shortcomings of supplementation/fortification, bio fortification” (breeding for higher mineral and vitamin content) of staple foods is a potential, practical, and effective approach for supplying nutrient-dense food to rural populations [39]. This strategy can complement existing initiatives by providing a long-term and less expensive means of eliminating under-nourished populations that rely on supplementation and commercial fortification for nutrition [40]. The bio fortification strategy entails one-time fixed costs for creating breeding procedures, breeding nutritional quality features into present crop varieties, and adapting these varieties to a variety of conditions [41]. After nutritious varieties have been disseminated, this technique will require minimal recurrent investments [42]. Furthermore, the expenditures do not rise in proportion to the number of people, and the advantages may be distributed globally, particularly to underdeveloped countries [43]. Finally, there will be no yield penalty if you breed for higher trace mineral density in the consumed plant portions [44]. The two approaches to bio fortifying crops with minerals such as iron and zinc include conventional and molecular breeding, as well as genetic engineering techniques [45]. Because polygene’s with small effects influence the uptake and accumulation of micronutrients in edible portions of plants, traditional breeding-based bio fortification efforts have had only limited effectiveness [46]. Furthermore, the success of this method is largely dependent on the natural variance in the gene pool [47]. Genetic engineering will be a viable option for enhancing micronutrients at targeted levels if there is insufficient genetic variability and fixable major gene effects [48]. Despite the enormous efforts made through traditional plant breeding programmes, utilizing the field of genetics (quantitative genetics, heterosis, transgressive segregants, mutational breeding, marker assisted breeding, QTL mapping, and so on) to entrap natural genetic variations for micronutrients and vitamin accumulation, there is still a long way to go: the dream of nourishing the world's population [49]. This is primarily due to a number of flaws and threats to conventional breeding, including the need for sufficient genetic variations for a trait in the species, which may not be available for many economically important crops, the need for genes targeting the trait in sexually compatible plants, the long time period required for breeding to introduce a single as well as multiple traits (pyramiding traits) into locally adapted elite varieties without the risk of linkage drag, and the lack of genes targeting the trait in sexually compatible plants (e.g. cereal seeds, tubers etc.) as well as the soil's reliance on phyto-availability of mineral nutrients [50]. Furthermore, the inverse link between grain yield and grain mineral concentration has made it difficult for traditional breeding approaches to overcome such trade-offs [51]. Genetic engineering to make transgenic has also been used to transfer genes directly into elite genotypes as a modern weapon to combat mineral deficit [52]. Transgenic technologies are methods that can be used to improve genotypes by altering certain metabolic pathways [53]. These technologies provide the door for the modification of proteins, vitamins, carbs, lipids, minerals, and other metabolites, which will be discussed in the sections that follow [54]. However, two factors should ideally be considered while developing transgenics for nutrient bio fortification: (a) selection of a widely adapted genotype of an economically important crop, (b) nutrient accumulation in the edible section of the crop plant without compromising plant physiology, development, or economic yield [55]. Bio fortification based on conventional breeding is a well-accepted strategy for improving micronutrients in crops [56]. Using conventional breeding, a large range of crops has been targeted for fortification [57]. When there is insufficient genotypic variation for the desired attribute within the species (e.g. provitamin A in rice), or when the crop is not amenable to conventional breeding, genetic engineering techniques offer a viable alternative to traditional breeding tactics (e.g. banana) [58]. New gene editing techniques such as transcription activator-like effect or nucleases and CRISPR/Cas9, as well as greater availability of fully sequenced genomes in staple crops, have opened up new possibilities for this bio fortification strategy [59]. Transgenes can be used to redistribute micronutrients between tissues, improve the efficiency of biochemical pathways in edible tissues, reconstruct selected pathways (for example, in the field of system biology), increase micronutrient bioavailability by removing anti nutrients, and transfer multiple genes in a single plant [60]. As a result, bridging the gap between plant breeders and molecular biologists is critical for harnessing the power of genetic alteration for agricultural plant bio fortification [61].

Literature Review

Transgenic approaches for improvement of iron, zinc and iodine concentrations

Because minerals are not produced in plants, they must obtain Fe and Zn from the rhizosphere and immediate environment [62]. Various crops have been genetically modified to increase mineral content, particularly Fe and Zn [63]. Transgenic techniques to boost Fe and Zn content in crops have primarily focused on modulating transporter expression in order to improve plant uptake and utilization efficiency [64]. Phytic acid, for example, is an anti-nutritional component that should be reduced. In contrast to Fe and Zn, transgenic methods [65]. In the sections that follow, we'll go through some of the Fe and Zn genes and transgenic techniques used in important grains [66]. (See Table 1)

Table 1. Biofortified transgenic crops with increased iron, zinc, and iodine.

Iron (Fe)

Rice, wheat, and maize are targeted in large bio fortification programmes to address micronutrient inadequacies since they provide more than half of the caloric need globally [67]. Lactoferrin (a Fe-chelating glycoprotein) and ferritin were used in experiments to raise the Fe content of the endosperm [68]. Lactoferrin is abundant in human milk (1-2 g/l) (LF). Created transgenic rice grains with the human LF gene under the control of the rice glutelin-1 promoter to boost Fe content for future use in newborn formula [69]. Heterologous protein expression was much higher than control, reaching 0.5 percent of grain weight, and bioavailability was validated using a human Caco-2 bioassay [70]. The hLF gene was expressed in transgenic japonica rice and that it accounted for about 1.5 percent of total soluble protein [71]. Ferritin, a localised protein found in plant plastids, is a significant non-toxic Fe storage form that can release Fe as needed for metabolic processes [72]. Ferritin is a widespread protein that stores around 4,500 Fe atoms in an accessible state [73]. As a result, increasing Fe accumulation through ferritin gene expression controlled by endosperm-specific promoters is an essential method for Fe bio fortification [74]. Overexpression of ferritin in numerous crops enhanced Fe content and bioavailability, according to studies produced rice transformants of Soyfer H1 under the endosperm-specific GluB1 rice promoter to boost Fe accumulation in endosperm of brown rice seeds, and showed a threefold increase in grain Fe content compared to non-transformed lines [75]. Under the control of the maize ubiquitin promoter, soybean ferritin cDNA was also transferred in wheat and rice [76]. Due to the substantial role of leaves as a sink, the resulting transformants had higher Fe content in leaves than seeds [77]. That extra ferritin sequesters Fe in the leaves, decreasing Fe mobilization to the seeds, is also a possibility [78]. It used a strong endosperm specific globulin promoter to introduce soybean ferritin into rice, resulting in a 13-fold increase in ferritin protein expression [79]. The Fe content, on the other hand, had just a modest enchantment (30%). These findings revealed that, in addition to increased Fe storage, higher Fe transport from the soil and greater translocation within the plant system are required [80]. Genetic engineering proved to be a promising technique for Fe bio fortification in cereals in the rice trials [81]. In comparison to rice, however, very little work has been done to enhance the Fe content of wheat and maize grains, particularly in the endosperm [82]. Cloned and analyzed wheat ferritin genes (TaFer1-A) and demonstrated that ferritin over expression in the endosperm of wheat can enhance Fe concentration. The TaFer1-A gene was over expressed in the endosperm under the control of the HMW glutein 1DX5 promoter, resulting in a 50-85 percent increase in wheat grain content [83].

 Iron-binding protein gene insertion

To generate Fe buildup in the seed that is ingested by humans, the first transgenic technique includes inserting an iron binding protein gene (lactoferrin) under the control of an endosperm specific promoter [84]. Plants or crops that store iron in their seeds are targeted for this purpose. Many attempts have been made to improve the grain iron content of rice, for example [84]. Rice is advantageous for a variety of reasons, including its low allergenicity and lack of harmful chemicals that interfere with gene expression [85]. Using a powerful endosperm specific promoter, researchers were able to successfully express human lactoferrin in de-husked rice. It resulted in a 120 percent increase in iron content, which was suitable for supplementing newborns; however, one molecule of lactoferrin binds to only two ferric ions, so it was still insufficient to fulfil the daily requirements of adults [86]. In wheat, soybean ferritin increased iron content by 1.5 and 1.9 times, respectively [87].

An iron-chelator gene was inserted

Nicotian Amine (NA) is an iron chelator that is important for Fe homeostasis and assimilation [88]. In rice (Table 1), the ferritin gene co-expressed with the Nicoti Anamine Synthase (NAS) gene results in a 6-fold rise in Fe content, which is higher than the single gene method [89]. In another study, expressing NAS, OsNAS3, resulted in a 7-fold increase in Fe content in transgenic rice seeds [90]. Anemic mice fed these transgenic rice seeds for four weeks recovered from iron deficiency and anaemia, whereas mice fed nontransgenic rice seeds showed no change. HvNAS1 over expressed by OsActin1 promoter/35S promoter in transgenic rice showed a 5-10 fold rise in endogenous NA levels in the shoots and seeds, resulting in a 3-fold larger increase in Fe content in T1 seeds. Similarly, transgenic tobacco with nicotianamine synthase (AtNAS1) overexpression had higher Fe levels. Bio fortified pearl millet has also been found to have a 2-fold increase in iron compared to most current wheat cultivars. An increase in iron absorption of 5-10% has been documented in roughly 35 million persons who eat bio fortified pearl millet [91].

Increased production of enhancers that improve Fe absorption

It's been proven that some foods can help with iron absorption. Vitamins including b-carotene, ascorbic acid, and α-tocopherol, as well as amino acids produced from proteins after digestion, fall into this category. Ascorbic acid and citric acid have been shown to decrease Fe to a ferrous form and increase small intestine absorption. As a result, transgenic techniques to over-express ascorbic acid in conjunction with ferritin can be applied. Fe absorption has also been demonstrated to benefit from higher cysteine concentration. Over expression of rice metallothionein-like protein resulted in a 10-fold increase in cysteine concentration in rice. It has 12 cysteine residues per mol of protein, identical to metallothionein. Golden rice with high levels of b-carotene has been found to have improved iron absorption.

Zinc (Zn)

Zn is a necessary element that also serves as a cofactor for over 300 enzymes and 1000 transcription factors. The natural variation of grain Zn content in cereals is modest. As a result, boosting the zinc content of cereal grains is critical for human nutrition and metabolism. Manipulation of the Zn content in cereal grains, on the other hand, may be more difficult than manipulating the Fe content found high connections between protein content, Fe, and Zn content in their study. Gpc-B1 (grain protein content b1) is a wheat quantitative trait locus linked to higher grain protein levels as well as higher Zn and Fe levels. After introducing the Gpc-B1 locus from the wild tetraploid wheat (Triticum turgidum ssp. Dicoccoides) into different recombinant chromosome substitution lines into cultivated wheat, an increase of 10–34 percent in grain Zn, Fe, Mn, and protein was observed, indicating the role of Gpc-B1 in the remobilization of protein, Zn, Fe, and Mn from the leaves to the grains. Over expression of genes involved in Zn translocation and mobilization resulted in improved Zn bioavailability without a yield penalty, which is an important strategy to improve grain Zn. Many cation transporters have been discovered in rice, but only a few have been studied in terms of substrate selectivity, expression pattern, and cellular localization. Members of the ZIP (ZRT, IRT-related protein) and CDF (Cation Diffusion Facilitator) families are the most common cation transporter families identified as being important in Zn uptake and translocation. In a thaliana root cells, the ZIP protein IRT1 plays an important role in Zn absorption. Introducing 35S enhancer elements to over-express NA synthase (NAS) resulted in 2-3 fold increases in Zn concentration in paddy. Similarly, polished rice grains from transgenic rice expressing the barley nicotianamine synthase gene HvNAS1 under the control of the rice actin1 promoter gathered 2–3 times more Zn. Thousands of IR64 and IR69428 transformants are created at IRRI using soybean or rice ferritin and rice nicotianamine synthase (NAS2) over expressed genetic constructs, and the Zn and Fe content in those lines has exceeded the goal level from field trials. As a result of the over expression of NAS genes, nicotianamine is a promising target for Zn bio fortification. Furthermore, bio fortifying grains using NAS alone or in conjunction with ferritin has a lot of promise in terms of addressing global human mineral deficiency. Sufficient research has been done in several crop species, including wheat; rice, maize, and barley, to better understand the Fe and Zn pathways in grain. Despite numerous obstacles such as the root-shoot barrier and grain filling, wheat researchers use rice-developed technologies and resources to boost Zn content in wheat grain, resulting in improved wheat lines.

NAS gene family over expression

Nicotianamine (NA) is a widespread chelator of transition metals (such as Zn and Fe) found in higher plants and is responsible for the transport of metal cations over short and long distances. The NA synthase (NAS) enzyme is involved in the production of NA by S-adenosylmethionine trimerization. Metal (Zn and Fe) profiles differentially control genes that encode for NAS in a range of plant species, including Arabidopsis, rice, maize, and barley. Over expression of exogenous or endogenous NAS genes is the most common method of increasing NA concentration in a plant using recombinant DNA technology. Exogenous HvNAS1 (barley NAS gene) over expression in tobacco and Arabidopsis has resulted in a several-fold rise in zinc, iron, and copper (Cu) concentrations in both plant species' seeds (Kim et al., 2005). In another study, over expression of HvNAS1 in rice resulted in a 15-fold rise in nicotianamine concentration (relative to wild type), as well as 1.5 and 2.5-fold increases in zinc and iron concentrations in polished rice grains. The rise in Fe and Zn concentration was caused by constitutive expression of AtNAS1 (Arabidopsis NAS gene) in combination with endosperm-specific production of ferritin. Furthermore, by expressing the OsNAS2 (endogenous NAS gene of rice) and Pv Ferritin (bean ferritin) genes in wheat grains, a significant level of Zn and Fe was attained. On the other hand, only a few researches have been published on the use of endogenous NAS to promote Planta NA expression for improved metal uptake in plants. Over expression of the endogenous NAS genes OsNAS1, OsNAS2, and OsNAS3 in rice resulted in significant increases in NA, Fe, and Zn concentrations in the endosperm of all three transgenic populations. In a different study, the OsNAS2 over expressing population had 20 and 2.7-fold higher NA and Zn concentrations than the wildtype population. Over expression of OsNAS3 resulted in a 9-fold increase in NA, a 2.2-fold increase in Zn, and a 2.6-fold increase in Fe concentration in polished rice grains.

NAC gene family over expression

Plant senescence is influenced by NAC transcription factors. Senescence is a synchronized process in which a whole plant or a portion of it remobilizes nutrients to younger tissues or seeds before dying. Senescence has been linked to increased Zn and Fe remobilization in studies. NAM-B1 (a member of the NAC transcription factors) has been discovered to play an important role in the early onset of senescence in wheat, which leads to a greater Zn concentration in grains. Reducing the amount of phytic acid in the body phytic acid (also known as phytate) is an inhibitor and antinutrient molecule that chelates minerals (Zn and Fe) and reduces their bioavailability, making it the most common cause of mineral deficiency in the globe. Because humans lack the intestinal phytase enzyme, phytic acid forms insoluble complexes with metal ions, particularly Zn and Fe, in the gastrointestinal system, which may not be absorbed or digested. By inhibiting their reabsorption into the body, phytate can form complexes with endogenously produced minerals, such as Zn. To counteract mineral deficiency, it is essential to reduce the amount of phytic acid in edible sections of staple crops to promote mineral absorption. Reduced phytate concentration in the diet has been linked to higher zinc absorption, according to research. Wheat transgenic lines had a 4-115 percent increase in bioavailable zinc in phytase.

Iodine

Iodine is required for the manufacture of the thyroid hormones triiodothyronine and thyroxine, making it a vital mineral for human health. Inadequate iodine intake affects more than 2 billion people worldwide. Triiodothyronine and thyroxine are two hormones that play an important role in metabolic control. Iodine shortage causes a reduction in the synthesis of these hormones, which leads to the growth of thyroid tissue, which is known as goiter. Goiter caused by iodine deficiency affects more than 187 million people worldwide. Furthermore, iodine deficiency during pregnancy may disrupt the offspring's neurodevelopment, whereas it affects somatic growth and cognitive skills during childhood. In comparison to other fortification techniques, adding iodine to table salt is a common strategy to prevent iodine deficiency. However, bio fortification of crops with iodine is a more promising strategy to combat mineral malnutrition because it is more sustainable and cost-effective. Several micronutrients, such as iron, zinc, and folate, have been successfully fortified into staple crops in recent decades. However, no reports of recombinant DNA-based bio fortification of iodine in crops have been published to yet. This is due to the physiology of iodine in plants being poorly understood. Iodine fluxes across the cell membrane of root cells via anion channels and probable H+/halides transporters, according to certain ideas. However, such transporters have yet to be found at the molecular level. Iodine volatilization from above-ground plant portions such as the root and leaf, on the other hand, is considerably well described in diverse species. Iodine volatilization is caused by the action of halide ion methyltransferase and halide/thiol methyltransferase. Transgenic Arabidopsis was the sole study that used genetic techniques to improve iodine concentration. The human sodium-iodide symporter (hNIS) gene of thyroid glands was over expressed under the CaMV 35S promoter in this study, resulting in increased iodine uptake. Furthermore, the HOL-1 gene, which encodes for the HMT enzyme (which causes iodine volatilization), was knocked out, and a significant reduction in volatilization was seen in transgenic plants compared to wild-type.

Conclusion

Micronutrients are crucial for human nutrition, particularly in the treatment of malnutrition in children and women. Bio fortification programmers rely heavily on micronutrients such as Fe, Zn and Iodine. Transgenic breeding is a strategic tool that can increase the content of these micronutrients in staple grains by several folds. Because genes for those desired qualities are available, it is conceivable to increase micronutrients in many staple crops. Trait-specific techniques have provided proof of concept for improving micronutrients through transgenic. It may be able to employ a combination of genes to improve micronutrients at the same time. After regulatory issues are resolved, transgenic crops will be widely planted to combat malnutrition. Furthermore, genome editing techniques for plant genes, such as CRISPR-Cas, ZFN, TALEN, and others, have recently showed significant promise in crop development. Crop bio fortification should also be made possible with genome editing techniques. Though genome editing-based bio fortification is still in its early stages, it should be widely used to accelerate bio fortification in cereals and horticulture crops, particularly vegetables. Bio fortified crop types with improved nutritional properties, on the other hand, must be tested in clinical studies for bioavailability and impact on end-user health.

Acknowledgement

Dr. Shambhoo Prasad Sir (Associate Professor), Department of Plant Molecular Biology & Genetic Engreening, Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya (UP) India, for his supervision, Thanks a lot in writing this review article and for the suggestions made.

References

1. Carvalho, Susana MP, and Vasconcelos Marta W. "Producing more with less: Strategies and Novel Technologies for Plant-Based Food Bio Fortification." Food Res Int 54 (2013): 961-971.

   [Crossref][Google Scholar]

2. Hoekenga, Owen A. "Genomics of Mineral Nutrient Biofortification: Calcium, Iron and Zinc." Geno Plant Genet Res (2014) 431-454.

   [Google Scholar]

3. World Health Organization (2000) Turning the Tide of Malnutrition: Responding to the Challenge of the 21st Century
4. Haseen, Farhana. "Malnutrition among Bangladeshi women in ultra-poor households: prevalence and determinants." Retrieved (2005)

   [Google Scholar]

5. BBS/UNICEF, Child and Mother Nutrition Survey of Bangladesh (CMNS) in 2005 Bangladesh Bureau of. Statistics and UNICEF, Dhaka, 2007.
6. Mannar MG, and Sankar R. "Micronutrient Fortification of Foods Rationale, Application and Impact." Indian J Pediatr 71 (2004): 997-1002.

   [Crossref][Google Scholar][Pubmed]

7. Genc, Yusuf, M Humphries Julia, H Lyons Graham, and D Graham Robin. "Exploiting Genotypic Variation in Plant Nutrient Accumulation to Alleviate Micronutrient Deficiency in Populations." J Trace Elem Med Biol 18 (2005): 319-324.

   [Crossref][Google Scholar][Pubmed]

8. Genç, Yusuf, Verbyla AP, Torun AA, and CakmakI, et al. "Quantitative trait loci analysis of zinc efficiency and grain zinc concentration in wheat using whole genome average interval mapping." Plant and Soil 314 (2009): 49-66.

   [Google Scholar]

9. Underwood EJ. Trace Elements in Human Nutrition, fourth ed., Academic, New York (1977) 545.
10. AS Prasad, (1978). Trace Elements and Iron in Human Metabolism,Wiley,NewYork/Chichester.
11. Singh, MV "Effect of trace element deficiencies in soil on human and animal health." Bull Indian Soc Soil Sci 27 (2009): 75-101.

    [Google Scholar]

12. Graham, Robin D, M Welch Ross, and E Bouis Howarth. "Addressing micronutrient malnutrition through enhancing the nutritional quality of staple foods: principles, perspectives and knowledge gaps." Adv Agron (2001): 77-142.

    [Crossref][Google Scholar]

13. White, Philip J, and R Broadley Martin. "Biofortification of Crops With Seven Mineral Elements Often Lacking in Human Dietsâ??Iron, Zinc, Copper, Calcium, Magnesium, Selenium and Iodine." New Phytologist 182 (2009): 49-84.

    [Crossref][Google Scholar][Pubmed]

14. Borrill, Philippa, Connorton James, Balk Janneke, and Tony Miller, et al. "Biofortification of Wheat Grain with Iron and Zinc: Integrating Novel Genomic Resources and Knowledge from Model Crops." Front Plant Sci 5 (2014): 53.

    [Crossref][Google Scholar][Pubmed]

15. Kumar, Sushil, Thirunavukkarasu Nepolean, Singh Govind, and Sharma Ramavtar, et al. "Biofortification for Selecting and Developing Crop Cultivars Denser in iron and zinc." Nutr Use Effi: Basics Advan (2015) 237-253.

    [Google Scholar].

16. Liu, Pam, Bhatia Rita, and Pachón Helena. "Food Fortification in India: A Literature Review." Indian J Community Heal 26 (2014): 59-74.

    [Google Scholar].

17. Naqvi, Shaista, Zhu Changfu, Farre Gemma, Ramessar Koreen, and Bassie Ludovic, et al. "Transgenic Multivitamin Corn through Biofortification of Endosperm with Three Vitamins Representing three Distinct Metabolic Pathways." Proce Natl Acad Sci 106 (2009): 7762-7767.

    [Crossref][Google Scholar][Pubmed]

18. Timmer, C Peter. "Biotechnology and food systems in developing countries." J Nutr 133 (2003): 3319-3322

    [Crossref][Google Scholar][Pubmed].

19. Bohra, Abhishek, Chand Jha Uday, and Kumar Sushil. "Enriching Nutrient Density in staple Crops Using Modern â??Omicsâ? tools." Biofortification Food Crops (2016) 85-103. 2016.

    [Google Scholar]

20. Cakmak, Ismail. "Enrichment of Cereal Grains with Zinc: Agronomic or GENETIC biofortification?." Plant Soil 302 (2008): 1-17.

    [Google Scholar].

21. Cakmak, Ä°smail, Torun AYFER, Millet E, and Feldman Moshe, et al. "Triticum dicoccoides: an important genetic resource for increasing zinc and iron concentration in modern cultivated wheat." Soil Sci Plant Nutrition 50 (2004): 1047-1054.

    [Crossref][Google Scholar]

22. Graham, Robin D, M Welch Ross, A Saunders David, and Ortiz�Monasterio Ivan et al. "Nutritious Subsistence Food Systems." Adv Agronomy 92 (2007): 1-74.

    [Crossref][Google Scholar]

23. Kumar, Sushil, T Hash Charles, Thirunavukkarasu Nepolean, and Singh Govind, et al. "Mapping Quantitative Trait Loci Controlling High Iron and Zinc Content in Self and Open Pollinated Grains of Pearl Millet [Pennisetum glaucum (L) R Br." Front Plant Sci 7 (2016): 1636.

    [Crossref][Google Scholar] [Pubmed].

24. Pfeiffer, Wolfgang H, and McClafferty Bonnie. "Harvestplus: Breeding Crops for Better Nutrition." Crop Sci 47 (2007): S-88.

    [Crossref][Google Scholar][Pubmed].

25. Grusak, Michael A, and DellaPenna Dean. "Improving the Nutrient Composition of Plants to Enhance Human Nutrition and Health." Ann Rev Plant Biol 50 (1999): 133-161.

    [Crossref][Google Scholar]

26. Johns, Timothy, and B Eyzaguirre Pablo. "Biofortification, biodiversity and diet: a search for complementary applications against poverty and malnutrition." Food Policy 32 (2007): 1-24.

    [Crossref][Google Scholar]

27. Kumar, Sushil, Tom Hash Charles, Nepolean Thirunavukkarasu, D Mahendrakar Mahesh, and Tara Satyavathi Chellapilla, et al. "Mapping Grain Iron and Zinc Content Quantitative Trait Loci in an Iniadi-Derived Immortal Population of Pearl Millet." Genes 9 (2018): 248.

    [Crossref][Google Scholar]

28. Tiwari, Vijay K, Rawat Nidhi, Chhuneja Parveen, and Neelam Kumari, et al. "Mapping Of Quantitative Trait Loci For Grain Iron And Zinc Concentration in Diploid A Genome Wheat." J Heredity 100 (2009): 771-776.

    [Crossref][Google Scholar] [Pubmed].

29. Bhullar, Navreet K, and Gruissem Wilhelm. "Nutritional enhancement of rice for human health: the contribution of biotechnology." Biotechnol Adv 31 (2013): 50-57

    [Crossref][Google Scholar] [Pubmed]

30. Dunwell, Jim M. "Transgenic Cereals: Current Status and Future Prospects." J Cereal Sci 59 (2014): 419-434.

    [Crossref][Google Scholar][Pubmed]

31. Garvin, David F, M Welch Ross, and W Finley John. "Historical Shifts in the Seed Mineral Micronutrient Concentration of US Hard Red Winter Wheat Germplasm." J Sci Food Agricuil 86 (2006): 2213-2220.

    [Crossref][Google Scholar]

32. SHI, Rong-li, Tong Yi-ping, Jing Rui-lian, and zhang Fu-suo, et al. "Characterization of Quantitative Trait Loci for Grain Minerals in Hexaploid Wheat (Triticum aestivum L.)." J Integrative Agricult 12 (2013): 1512-1521.

    [Crossref][Google Scholar]

33. Fan, Ming-Sheng, Zhao Fang-Jie, Fairweather-Tait Susan J, and R Poulton Paul, et al. "Evidence of Decreasing Mineral Density in Wheat Grain Over the Last 160 years." J Trace Elem Med Biol 22 (2008): 315-324.

    [Crossref][Google Scholar][Pubmed]

34. Oury, F-X, Leenhardt Fanny, Remesy C, and Chanliaud E, et al. "Genetic variability and stability of grain magnesium, zinc and iron concentrations in bread wheat." Europ J Agronom 25 (2006): 177-185.

    [Crossref][Google Scholar]

35. Wenefrida, Ida, S Utomo Herry, and D Linscombe Steve. "Mutational Breeding and Genetic Engineering in the Development of High Grain Protein Content." J Agric Food Chem 61 (2013): 11702-11710.

    [Crossref][Google Scholar][Indexed at]

36. Vanderschuren, Herve, Svetlana Boycheva, Kuan-Te Li, and Nicolas Szydlowski, et al. "Strategies for vitamin B6 biofortification of plants: a dual role as a micronutrient and a stress protectant." Front Plant Sci 4 (2013): 143.

    [Crossref][Google Scholar][Pubmed]

37. Garg, Monika, Natasha Sharma, Saloni Sharma, and Payal Kapoor, et al. "Biofortified crops generated by breeding, agronomy, and transgenic approaches are improving lives of millions of people around the world." Front Nutri (2018): 12.

    [Crossref][Google Scholar][Pubmed]

38. Biofortification Progress Briefs, 2014. http://www.harves tplus.org/sites/def ault/files/ Biofortification_Progress_Briefs_August2014_WEB_2.pdf.
39. Ricroch, Agnes, Pauline Clairand, and Wendy Harwood. "Use of CRISPR systems in plant genome editing: toward new opportunities in agriculture." Emerg Topics Life Sci 1 (2017): 169-182.

    [Crossref][Google Scholar][Pubmed]

40. Morrissey, Joe, and Mary Lou Guerinot. "Iron uptake and transport in plants: the good, the bad, and the ionome." Chemical Rev 109 (2009): 4553-4567.

    [Crossref][Google Scholar][Pubmed]

41. Kerkeb, Loubna, Indrani Mukherjee, Iera Chatterjee, and Brett Lahner, et al. "Iron-induced turnover of the Arabidopsis IRON-REGULATED TRANSPORTER1 metal transporter requires lysine residues." Plant Physiol 146 (2008): 1964-1973.

    [Crossref][Google Scholar][Pubmed]

42. Masuda, Hiroshi, Kanako Usuda, Takanori Kobayashi, and Yasuhiro Ishimaru, et al. "Overexpression of the barley nicotianamine synthase gene HvNAS1 increases iron and zinc concentrations in rice grains." Rice 2 (2009): 155-166.

    [Crossref][Google Scholar][Indexed]

43. Brinch-Pedersen, Henrik, Soren Borg, Birgitte Tauris, and Preben B Holm, et al. "Molecular genetic approaches to increasing mineral availability and vitamin content of cereals." J Cereal Sci 46 (2007): 308-326.

    [Crossref][Google Scholar][Indexed]

44. Palmgren, Michael G, Stephan Clemens, Lorraine E Williams, and Ute Kramer, et al. "Zinc biofortification of cereals: problems and solutions." Trends Plant Sci 13 (2008): 464-473.[Crossref]

    [Google Scholar][Pubmed]

45. Sharma, Ashish, Deepti Shankhdhar, and Shailesh Chandra Shankhdhar. "Plant growth promoting rhizobacteriaâ??an approach for biofortification in cereal grains." Physiol Efficiency Crop Improvment (2015): 460-545.

    [Google Scholar][Indexed]

46. Ozturk, Levent, Mustafa Atilla Yazici, Cemal Yucel, and Ayfer Torun, et al. Concentration and localization of zinc during seed development and germination in wheat." Physiologia plantarum 128 (2006): 144-152.

    [Crossref][Google Scholar][Indexed]

47. Uauy, Cristobal, Juan Carlos Brevis, and Jorge Dubcovsky. "The high grain protein content gene Gpc-B1 accelerates senescence and has pleiotropic effects on protein content in wheat." JExperimen Bot 57 (2006): 2785-2794.

    [Crossref][Google Scholar][Pubmed]

48. Distelfeld, Assaf, Ismail Cakmak, Zvi Peleg, and Levent Ozturk, et al. "Multiple QTL�effects of wheat Gpc�B1 locus on grain protein and micronutrient concentrations." Physiologia Plantarum 129 (2007): 635-643.

    [Crossref][Google Scholar][Indexed]

49. Korshunova, Yulia O, David Eide, W Gregg Clark, and Mary Lou Guerinot, et al. "The IRT1 protein from Arabidopsis thaliana is a metal transporter with a broad substrate range." Plant Mole Biol 40 (1999): 37-44.

    [Crossref][Google Scholar][Pubmed]

50. Lee, Sichul, Un Sil Jeon, Seung Jin Lee, and Yoon-Keun Kim, et al. "Iron fortification of rice seeds through activation of the nicotianamine synthase gene." Proceed Natl Acad Sci 106 (2009): 22014-22019.

    [Crossref][Google Scholar][Indexed]

51. Zheng, Luqing, Zhiqiang Cheng, Chunxiang Ai, and Xinhang Jiang, et al. "Nicotianamine, a novel enhancer of rice iron bioavailability to humans." PLoS One 5 (2010): e10190.

    [Crossref][Google Scholar][Pubmed]

52. Masuda, Hiroshi, Yasuhiro Ishimaru, May Sann Aung, and Takanori Kobayashi, et al. "Iron biofortification in rice by the introduction of multiple genes involved in iron nutrition." Scientific Rep 2 (2012): 1-7.

    [Crossref][Google Scholar][Pubmed]

53. Brinch-Pedersen, Henrik, Soren Borg, Birgitte Tauris, and Preben B Holm, et al. "Molecular genetic approaches to increasing mineral availability and vitamin content of cereals." J Cereal Sci46 (2007): 308-326.

    [Crossref][Google Scholar][Indexed]

54. Kanyshkova, T.G., Buneva, V.N., Nevinsky, G.A. (2001). "Lactoferrin and its biological functions". Biochem 66: 1-7.

    [Crossref][Google Scholar][Pubmed]

55. Nandi, Somen, Yasushi A Suzuki, Jianmin Huang, and Doris Yalda, et al. "Expression of human lactoferrin in transgenic rice grains for the application in infant formula." Plant Sci 163 (2002): 713-722.[Crossref]

    [Google Scholar][Indexed]

56. Lee, Jin Hyoung, Hyo Sung Kim, Seok Cheol Suh, and Seong Lyul Rhim, et al. "Development of transgenic rice lines expressing the human lactoferrin gene." J Plant Biotechnol 37 (2010): 556.

    [Crossref][Google Scholar][Indexed]

57. Darbani, Behrooz, Jean-Francois Briat, Preben Bach Holm, and Soren Husted, et al. "Dissecting plant iron homeostasis under short and long-term iron fluctuations." Biotechnol Advan 31 (2013): 1292-1307.

    [Crossref][Google Scholar][Pubmed]

58. Drakakaki, Georgia, Sylvain Marcel, Raymond P Glahn, and Elizabeth K Lund, et al. "Endosperm-specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron." Plant Mole Biol 59 (2005): 869-880.

    [Crossref][Google Scholar][Pubmed]

59. Lucca, Paola, Richard Hurrell, and Ingo Potrykus. "Fighting iron deficiency anemia with iron-rich rice." J Ame Coll Nutr 21(2002): 184S-190S.

    [Google Scholar][Pubmed][Indexed]

60. Vasconcelos, Marta, Karabi Datta, Norman Oliva, and Mohammad Khalekuzzaman, et al. "Enhanced iron and zinc accumulation in transgenic rice with the ferritin gene." Plant Sci 164 (2003): 371-378.

    [Crossref][Google Scholar][Indexed]

61. Qu, Le Qing, Toshihiro Yoshihara, Akio Ooyama, and Fumiyuki Goto, et al. "Iron accumulation does not parallel the high expression level of ferritin in transgenic rice seeds." Planta 222 (2005): 225-233.

    [Crossref][Google Scholar][Pubmed]

62. Drakakaki, Georgia, Sylvain Marcel, Raymond P Glahn, and Elizabeth K Lund, et al. "Endosperm-specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron." Plant Mole Biol59 (2005): 869-880.[Crossref]

    [Google Scholar][Pubmed]

63. Lucca, Paola, Richard Hurrell, and Ingo Potrykus. "Fighting iron deficiency anemia with iron-rich rice." J Ame College Nutrition 21(2002): 184S-190S.

    [Google Scholar][Pubmed][Indexed]

64. Vasconcelos, Marta, Karabi Datta, Norman Oliva, and Mohammad Khalekuzzaman, et al. "Enhanced iron and zinc accumulation in transgenic rice with the ferritin gene." Plant Sci164 (2003): 371-378.

    [Crossref][Google Scholar][Indexed]

65. Qu, Le Qing, Toshihiro Yoshihara, Akio Ooyama, and Fumiyuki Goto, et al. "Iron accumulation does not parallel the high expression level of ferritin in transgenic rice seeds." Planta 222 (2005): 225-233.

    [Crossref][Google Scholar][Pubmed]

66. Aluru, Maneesha R., Steve R. Rodermel, and Manju B Reddy. "Genetic modification of low phytic acid 1-1 maize to enhance iron content and bioavailability." J Agri Food Chem 59 (2011): 12954-12962.

    [Crossref][Google Scholar][Pubmed]

67. Drakakaki, Georgia, Paul Christou, and Eva Stoger. "Constitutive expression of soybean ferritin cDNA intransgenic wheat and rice results in increased iron levels in vegetative tissues but not in seeds." Transgenic Res 9 (2000): 445-452.

    [Crossref][Google Scholar][Pubmed]

68. Borg, Soren, Henrik Brinch-Pedersen, Birgitte Tauris, and Lene Heegaard Madsen, et al. "Wheat ferritins: improving the iron content of the wheat grain." J Cereal Sci 56 (2012): 204-213.

    [Crossref][Google Scholar][Indexed]

69. Goto, Fumiyuki, Toshihiro Yoshihara, Naoki Shigemoto, and Seiichi Toki, et al. "Iron fortification of rice seed by the soybean ferritin gene." Nature biotechnol 17 (1999): 282-286.

    [Crossref][Google Scholar][Pubmed]

70. Lucca, Paola, Richard Hurrell, and Ingo Potrykus. "Genetic engineering approaches to improve the bioavailability and the level of iron in rice grains." Theoret Appl Genetics 102 (2001): 392-397.

    [Crossref][Google Scholar][Indexed]

71. Masuda, Hiroshi, Kanako Usuda, Takanori Kobayashi, and Yasuhiro Ishimaru, et al. "Overexpression of the barley nicotianamine synthase gene HvNAS1 increases iron and zinc concentrations in rice grains." Rice 2 (2009): 155-166.

    [Crossref][Google Scholar][Indexed]

72. Lucca, Paola, Richard Hurrell, and Ingo Potrykus. "Approaches to improving the bioavailability and level of iron in rice seeds." J Sci Food Agri 81 (2001): 828-834.

    [Crossref][Google Scholar][Indexed]

73. Ishimaru, Yasuhiro, Hiroshi Masuda, Khurram Bashir, and Haruhiko Inoue, et al. "Rice metal�nicotianamine transporter, OsYSL2, is required for the long distance transport of iron and manganese." Plant J 62 (2010): 379-390.

    [Crossref][Google Scholar][Pubmed]

74. Kobayashi, Takanori, Reiko Nakanishi Itai, Yuko Ogo, and Yusuke Kakei, et al. "The rice transcription factor IDEF1 is essential for the early response to iron deficiency, and induces vegetative expression of late embryogenesis abundant genes." Plant J 60 (2009): 948-961.

    [Crossref][Google Scholar][Pubmed]

75. Suzuki, Yasushi A, Kouichirou Shin, and Bo Lonnerdal. "Molecular cloning and functional expression of a human intestinal lactoferrin receptor." Biochem 40 (2001): 15771-15779.

    [Crossref][Google Scholar][Pubmed]

76. Ricachenevsky, Felipe K, Paloma K Menguer, Raul A Sperotto, and Lorraine E Williams, et al. "Roles of plant metal tolerance proteins (MTP) in metal storage and potential use in biofortification strategies." Front Plant Sci 4 (2013): 144.

    [Crossref][Google Scholar][Pubmed]

77. Singh, Simrat Pal, Beat Keller, Wilhelm Gruissem, and Navreet K Bhullar, et al. "Rice NICOTIANAMINE SYNTHASE 2 expression improves dietary iron and zinc levels in wheat." Theoretical Appl Genetics 130 (2017): 283-292.

    [Crossref][Google Scholar][Pubmed]

78. Brinch-Pedersen, Henrik, Annette Olesen, Soren K Rasmussen, and Preben B Holm, et al. "Generation of transgenic wheat (Triticum aestivum L.) for constitutive accumulation of an Aspergillus phytase." Mole Breeding 6 (2000): 195-206.

79. [Crossref][Google Scholar][Indexed]

80. Borg, Soren, Henrik Brinch-Pedersen, Birgitte Tauris, and Lene Heegaard Madsen, et al. "Wheat ferritins: improving the iron content of the wheat grain." J Cereal Sci 56 (2012): 204-213.

    [Crossref][Google Scholar][Indexed]

81. Connorton, James M, Eleanor R Jones, Ildefonso Rodriguez-Ramiro, and Susan Fairweather-Tait, et al. "Wheat vacuolar iron transporter TaVIT2 transports Fe and Mn and is effective for biofortification." Plant Physiol 174 (2017): 2434-2444.

    [Crossref][Google Scholar][Pubmed]

82. Xiaoyan, Sui, Zhao Yan, and Wang Shubin. "Improvement Fe content of wheat (Triticum aestivum) grain by soybean ferritin expression cassette without vector backbone sequence." J Agri Biotechnol (2012).

    [Google Scholar]

83. Gao, Xiao Rong, Guo Kun Wang, Qiao Su, and Yan Wang, et al. "Phytase expression in transgenic soybeans: stable transformation with a vector-less construct." Biotechnol letters 29 (2007): 1781-1787.

    [Crossref][Google Scholar][Pubmed]

84. Ramesh, Sunita A, Steve Choimes, and Daniel P Schachtman. "Over-expression of an Arabidopsis zinc transporter in Hordeum vulgare increases short-term zinc uptake after zinc deprivation and seed zinc content." Plant mole biol 54 (2004): 373-385.

    [Crossref][Google Scholar][Indexed]

85. Drakakaki, Georgia, Sylvain Marcel, Raymond P Glahn, and Elizabeth K Lund, et al. "Endosperm-specific co-expression of recombinant soybean ferritin and Aspergillus phytase in maize results in significant increases in the levels of bioavailable iron." Plant Mole Biol 59 (2005): 869-880.

    [Crossref][Google Scholar][Pubmed]

86. Rogers, Elizabeth E, and Mary Lou Guerinot. "FRD3, a member of the multidrug and toxin efflux family, controls iron deficiency responses in Arabidopsis." Plant Cell 14 (2002): 1787-1799.

    [Crossref][Google Scholar][Pubmed]

87. Douchkov, D, C Gryczka, UW Stephan, and R Hell, et al. "Ectopic expression of nicotianamine synthase genes results in improved iron accumulation and increased nickel tolerance in transgenic tobacco." Plant, Cell Environ 28 (2005): 365-374.

    [Crossref][Google Scholar][Indexed]

88. Lanquar, Viviane, Francoise Lelievre, Susanne Bolte, and Cecile Hames, et al. Carine Alcon, Dieter Neumann, Gérard Vansuyt et al. "Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron." The EMBO J 24 (2005): 4041-4051.

    [Crossref][Google Scholar][Pubmed]

89. Halka, Mariya, Sylwester Smolen, MaÅ?gorzata Czernicka, and Magdalena Klimek-Chodacka, et al. "Iodine biofortification through expression of HMT, SAMT and S3H genes in Solanum lycopersicum L." Plant Physiol Biochem 144 (2019): 35-48.

    [Crossref][Google Scholar][Pubmed]

90. Lee, Sichul, Yu-Young Kim, Youngsook Lee, and Gynheung An, et al. "Rice P1B-type heavy-metal ATPase, OsHMA9, is a metal efflux protein." Plant Physiol 145 (2007): 831-842.

    [Crossref][Google Scholar][Pubmed]

91. Connorton, James M, Eleanor R Jones, Ildefonso Rodriguez-Ramiro, and Susan Fairweather-Tait, et al. "Wheat vacuolar iron transporter TaVIT2 transports Fe and Mn and is effective for biofortification." Plant Physiol 174 (2017): 2434-2444.

    [Crossref][Google Scholar][Pubmed].