Fnut 08 721728

REVIEW
published: 07 October 2021

doi: 10.3389/fnut.2021.721728
Biofortification of Cereals and Pulses

Using New Breeding Techniques:
Current and Future Perspectives
Rahil Shahzad 1*† , Shakra Jamil 1† , Shakeel Ahmad 2*† , Amina Nisar 3 , Sipper Khan 4 ,
Zarmaha Amina 4 , Shamsa Kanwal 1 , Hafiz Muhammad Usman Aslam 5 , Rafaqat Ali Gill 6
and Weijun Zhou 7*
1
Agricultural Biotechnology Research Institute, Ayub Agricultural Research Institute, Faisalabad, Pakistan, 2 Maize Research
Edited by:
Station, Ayub Agricultural Research Institute, Faisalabad, Pakistan, 3 Department of Plant Breeding and Genetics, University
Marcello Iriti,
of Agriculture, Faisalabad, Pakistan, 4 Tropics and Subtropics Group, Institute of Agricultural Engineering, University of
University of Milan, Italy
Hohenheim, Stuttgart, Germany, 5 Institute of Plant Protection, Muhammad Nawaz Sharif University of Agriculture, Multan,
Reviewed by: Pakistan, 6 Key Laboratory of Biology and Genetic Improvement of Oil Crops, The Ministry of Agriculture and Rural Affairs, Oil
Simon Gerben Strobbe, Crops Research Institute of Chinese Academy of Agricultural Sciences, Wuhan, China, 7 Key Laboratory of Spectroscopy
Ghent University, Belgium Sensing, The Ministry of Agriculture and Rural Affairs, Institute of Crop Science, Zhejiang University, Hangzhou, China
Jitendra Kumar,
Indian Institute of Pulses Research
(ICAR), India Cereals and pulses are consumed as a staple food in low-income countries for the
*Correspondence: fulfillment of daily dietary requirements and as a source of micronutrients. However, they
Rahil Shahzad are failing to offer balanced nutrition due to deficiencies of some essential compounds,
rahilshahzad91@gmail.com
Shakeel Ahmad macronutrients, and micronutrients, i.e., cereals are deficient in iron, zinc, some essential
shakeelpbg@gmail.com amino acids, and quality proteins. Meanwhile, the pulses are rich in anti-nutrient
Weijun Zhou
wjzhou@zju.edu.cn
compounds that restrict the bioavailability of micronutrients. As a result, the population
is suffering from malnutrition and resultantly different diseases, i.e., anemia, beriberi,
† These authors have contributed
equally to this work
pellagra, night blindness, rickets, and scurvy are common in the society. These facts
highlight the need for the biofortification of cereals and pulses for the provision of
Specialty section: balanced diets to masses and reduction of malnutrition. Biofortification of crops may
This article was submitted to
be achieved through conventional approaches or new breeding techniques (NBTs).
Nutrition and Sustainable Diets,
a section of the journal Conventional approaches for biofortification cover mineral fertilization through foliar or soil
Frontiers in Nutrition application, microbe-mediated enhanced uptake of nutrients, and conventional crossing
Received: 07 June 2021 of plants to obtain the desired combination of genes for balanced nutrient uptake and
Accepted: 23 August 2021
Published: 07 October 2021
bioavailability. Whereas, NBTs rely on gene silencing, gene editing, overexpression, and
Citation:
gene transfer from other species for the acquisition of balanced nutritional profiles in
Shahzad R, Jamil S, Ahmad S, mutant plants. Thus, we have highlighted the significance of conventional and NBTs
Nisar A, Khan S, Amina Z, Kanwal S,
for the biofortification of cereals and pulses. Current and future perspectives and
Aslam HMU, Gill RA and Zhou W
(2021) Biofortification of Cereals and opportunities are also discussed. Further, the regulatory aspects of newly developed
Pulses Using New Breeding biofortified transgenic and/or non-transgenic crop varieties via NBTs are also presented.
Techniques: Current and Future
Perspectives. Front. Nutr. 8:721728. Keywords: anti-nutrients, CRISPR-Cas, conventional breeding, fertilization, malnutrition, micronutrients, genome
doi: 10.3389/fnut.2021.721728 editing, transgenic breeding
Frontiers in Nutrition | www.frontiersin.org 1 October 2021 | Volume 8 | Article 721728

Shahzad et al. Biofortification of Crops Using NBTs
INTRODUCTION
BOX 1 | Terms and definitions related to biofortification of crops.
The products of cereal and pulses (Box 1) are used as a staple
food in developing and developed countries and serve as a Cereals: A cereal is any grass cultivated for edible components of its grain,
which is composed of germ, endosperm, and bran.
source of nutrients and dietary energy. On average, cereals Pulses: Pulses are a type of leguminous crop that are harvested solely
are composed of ∼75% carbohydrates (mainly starch), 6– for the dry seed. Dried beans, lentils, and peas are the most known and
15% proteins in full-grain, which may vary from species to consumed types of pulses. Pulses do not include crops that are harvested
species, and contribute ∼50% of the global energy terms. The green (e.g., green peas, green beans).
Biofortification: It is a process of increasing the nutritive value of a food
importance of cereals can be judged from the fact that global
crop through use of fertilizer, selective breeding, or genetic modification.
food security to a greater degree depends on their availability Macronutrient: Macronutrients could be defined as chemical elements or a
which amounts to 2,600 million tons annually (1). The changing class of chemical compounds that are consumed in large quantities by the
climate is putting heavy pressure on crop production with the human body for the sake of energy for growth, metabolism, and other body
increasing demand for crops that can withstand harsh climatic functions.
Micronutrient: Micronutrients include dietary minerals and vitamins that
conditions, i.e., drought and heat stress, and can deliver a
are required in very small quantities (<100 mg per day) and not involved in
balanced diet to human beings (2, 3). Under these circumstances, regulation of growth directly. However, these are vital for health development,
pulses have emerged as an important component of the food disease prevention, and well-being.
chain, which can provide an environmentally stable source Malnutrition: Malnutrition is a condition that results from eating a diet which
of protein, fats, and micronutrients (Box 1). Pulses are great does not supply a healthy amount of one or more nutrient. These include
diets that either contain too much nutrient or not enough nutrient.
sources of complex carbohydrates, dietary proteins, minerals, Over-nutrition: It is a form of malnutrition that arises due to intake of a diet
and vitamins for human nutrition. These are excessively used in having insufficient energy and nutrient amount.
various parts of the world as traditional diets because of high Hidden hunger: It describes a state of deficiency of essential vitamins and
protein concentration, balanced amino acid profiling, and slow minerals in the human diet.
Food fortification: It is the process of adding micronutrients to the food
digestibility of carbohydrates. These are popularly used because
with the aim of delivering a balanced diet.
of the delivery of proteins and micronutrients and balanced diets Dietary diversification: It is a process of changing the dietary preferences
to the masses in a cost-efficient manner. These along with cereals of the household, i.e., increasing the uptake of animal-sourced food.
offer a complete diet if biofortified for nutrient compounds Supplementation: It is a term used to describe the provision or relatively
(4, 5). large amount of micronutrients in the form of pills, tablets, capsules, or
syrups to improve nutrition health in the short term.
The United Nations (UNs) has set 17 sustainable development Agronomic biofortification: It describe the biofortification method in which
goals (SDGs) in 2015. Out of which, SDG3 is about “ensuring deliberate application of mineral fertilization is carried out to increase the
healthy lives and promote well-being for all at all ages.” Good concentration of the desired micronutrients in the edible part of the crop to
health starts with nutrition; however, without regular and increase dietary intake.
nutritious food humans cannot live, learn, fend off diseases, Conventional breeding: It is a process of development of new varieties of
or lead productive lives (https://sdgs.un.org/goals/goal3). crops by using older techniques and natural processes without using the
latest molecular plant biological tools.
According to the European Food Safety Authority report, the New breeding techniques (NBTs): These are crop improvement
daily dietary reference values of several nutrient elements for techniques that make specific changes with the plant DNA in order to
adults are 8–11 mg for zinc, 8–18 mg for iron, and 750 mg for change the trait of interest. These modifications may vary from single base
calcium, depending on gender, which is not met in our daily diets, pair addition, deletion, substitution to removal, or addition of complete gene
in an organism.
leading to micronutrient deficiency (6). Micronutrient deficiency
Transgenic breeding: It refers to the genetic improvement of crop plants
is a silent epidemic—it slowly weakens the immune system, in relation to various economic traits useful for human beings by means of
stunting physical and intellectual growth, and even causing death genetic engineering tools.
(7). Micronutrient deficiency, also known as hidden hunger Genetic engineering: It is a process of using the recombinant DNA
(Box 1), is extremely pronounced with more than 2 billion technology to alter the genetic make-up of an organism.
RNA interference: This term is used to describe a cellular mechanism that
masses affected by it (8). This deficiency escalates the probability uses a gene’s own DNA sequence to turn it off in a process called gene
of infectious diseases and deaths resulting from diarrhea, measles, silencing. In plants, animals, and fungi, RNAi is triggered by double-stranded
malaria, and pneumonia in numerous low-income countries. RNA (dsRNA).
Food fortification (Box 1) through supplementation and crop Genome editing: It is a crop improvement technique in which DNA is
biofortification is considered as an industrialized solution inserted, deleted, modified, or replaced in the genome at a particular location
without disturbing the rest of the genome.
for this alarming nutritional deficiency (9). Biofortification Transgenic crops: Transgenic or genetically modified crops are plants that
(Box 1) is a micronutrient-enriching approach, involving have DNA modified using genetic engineering methods.
strategies focused on targeting and modulation of movement Biofortified crops: These are described as nutritionally enhanced food
pathways, i.e., root uptake phenomenon, transporting, crops offering increased bioavailability of different nutrients to the human
population.
remobilizing, storage, and increased bioavailability of the
Overexpression: It is a process of making too many copies of a plant
minerals. The four main strategies widely employed in crops’ protein by attaching an upstream constitutive promotor using genetic
biofortification include agronomic biofortification focused on engineering tools.
better mineral solubilizing and mobilization while conventional (Continued)
plant breeding, genetic engineering, and gene editing mainly

as well as availability of sequence genome of multiple species

BOX 1 | Continued opened new horizons for crop biofortification (13, 14). Because of
the above-mentioned facts, we have exploited the potential of new
Mega-nucleases: These are characterized by a large recognition site
(double-stranded DNA sequences of 12–40 base pairs) which gives them an
breeding techniques (NBTs), i.e., genetic engineering, RNAi, and
advantage of having only a unique target site once in a genome to achieve gene editing for the development of nutritionally enriched crops
target specificity. These are equally effective for modification of genomes of to combat malnutrition (Box 1). Further regulatory aspects of
all organisms. crops developed through NBT and prospects were also discussed
Zinc finger nucleases (ZFNs): These are artificially designed nucleases
in light of the latest developments.
which are comprised of zinc finger DNA binding domain attached to a DNA
cleavage domain. Zinc finger domain can be engineered to target specific
target DNA sequences; resultantly, ZFNs target a specific gene in a complex
genome of large organisms to achieve target specificity. MALNUTRITION—A HIDDEN HUNGER
Transcription activator-like effector nuclease (TALENs): These are
restrictions enzymes that can be engineered to cut DNA at a specific target
Malnutrition is an emerging threat in an ever-increasing
site. These are also comprised of two domains, i.e., TAL effector binding
DNA domain and DNA cleavage domain. population around the globe. The world population is expected
Clustered regularly interspaced short palindromic repeats to rise to 8.3 billion in 2030 against the present-day 7.8 billion.
(CRISPR)/Cas9: It is the simplest, most versatile, and precise method of According to UNFAO, 792.5 million people around the globe
genome editing which is comprised of two components, i.e., Cas9 enzyme suffer from malnutrition with more victims in the developing
which acts as a pair of molecular scissors which can cut the two strands of
world. Most people in developing countries either face hunger
DNA at a specific location in the genome which can be added or removed
and guide-RNA (gRNA) which is a small piece of predesigned RNA sequence or receive nutrient-poor food. The global death rate due to
(about 20 bases long) located inside a longer RNA scaffold. Upon binding of hunger piles up to 24,000 people per day (15). About one-
the scaffold to the target DNA, gRNA guide Cas9 and make sure that the third of the population is suffering from “hidden hunger.” They
cleavage takes place at the target site. lack either one or all of the essential micro- or macronutrients
Base editing: It is a CRISPR based gene editing system which offers
introduction of point mutations at the desired target site without generating
(Box 1), like Zn, Fe, selenium, iodine, folic acid, lysine, vitamin
double stranded breaks. Two major classes of base editors have been A, vitamin B12, vitamin C, vitamin D, and others (Table 1), in
identified, i.e., cytidine base editors (CBE) which allow C>T conversion and their diets (12, 33). These vitamins and nutrients are inevitable
adenine base editors (ABE) which allow A>G conversions. for the growth, development, and proper functioning of the
Prime editing (PE): It is a search and replace genome editing tool in which
human body. Deficiency of these micronutrients for a short
pegRNA complex binds to the target DNA and Cas9 nicks only one strand
generating a flap and allowing donner free precise genome editing. PE not
period does not cause much harm but their shortage for a
only allows all sorts of transitions and transversions, but also allows small long time may cause many diseases like anemia (Fe deficiency),
insertions and deletions. beriberi (Vitamin B deficiency), pellagra (niacin deficiency),
Anti-nutrients compounds: These are natural or synthetic compounds rickets (vitamin D deficiency), and scurvy (vitamin C deficiency)
that interfere with the adsorption of essential nutrients and make them
or be fatal (Table 1) (34, 35). Approximately 25% of the world’s
unavailable to human beings, e.g., phytate, lectins, tannins, protease
inhibitors, and calcium oxalate. population suffers from anemia and Fe deficiency is its leading
cause (50–60% affected). Beriberi is responsible for 50% of the
deaths of children in the early 1920s in rice-consuming regions
around the globe (36). Rickets is a very serious threat to many
communities around the globe, i.e., China has 3%, Bangladesh
focus on cultivar improvement in terms of micronutrient 13%, Magnolia 25.6%, India 13%, and the Middle East 10%
accumulation, bioavailability, and suppression of anti-nutrients population affected by this disease (37). These facts highlight the
(Box 1) (10). importance of biofortification of crops to deliver healthy diets to
Biofortification was initially designed to overcome the the world’s population to overcome these issues.
drawbacks and deficiencies of supplementation (11). Therefore, a Most people depend on cereals and pulses to fulfill their
modern weapon against mineral deficiency involved transferring dietary requirements. Around 60% of the calories and dietary
the genes directly in elite genotypes. Transgenic crops employed requirements in the developing world and 30% in the developed
the genetic engineering tools for genotype improvement in world are extracted from cereals- and pulses-based food (38).
focused metabolic pathways of the plant to improve and However, cereal does not have a balanced nutritional profile,
modify carbohydrates, fats, proteins, minerals, vitamins, and i.e., iron and zinc and other essential nutrients, which is
other secondary metabolites (12). These transgenes targeted the leading cause of malnutrition (Table 1). The population
redistribution of micronutrients between tissues, enhanced the residing in developing countries is at high risk of malnutrition;
efficiency of a biochemical pathway, increased bioavailability, more than 1 in 5 newborns did not receive a balanced diet
reduced anti-nutrient absorption and multigene transfer (corn and suffer from impaired cognitive development. More than
with a high concentration of beta-carotene, ascorbate, and 40% of children in South Asia and Africa are suffering from
folate in a multivitamin plant) with the reconstruction of malnutrition. Approximately 1 in 14 children are wasted, 1 in
selected pathways (field of system biology) (12). With the 20 children are overweight, and 45% of deaths in <5-year-old
advent of various “Omics” technologies, different CRISPR- children are associated with undertaking of a nutrient-poor diet.
based gene-editing tools including CRISPR-Cas9/13 (Box 1) and These facts are associated with undertaking a nutrient poor diet
transcription activator-like effector nucleases (TALENs) (Box 1) by a newborn and its lactating mother (39, 40). Half of the

TABLE 1 | Essential micro- and macro-nutrients required for good human health.
Nutrient Composition in Quantity in pulses Deficiency symptoms in humans References

cereals
Micronutrients (mg/100 g food)

Iron (Fe) 0.500–3.800 1.250–3.330 Anemia (16, 17)
Zinc (Zn) 0.600–3.300 1.060–1.530 Wilson’s disease, sickle cell disease, chronic kidney disease, chronic (16, 17)
liver disease
Copper (Cu) 0.001–0.012 0.180–0.290 Anemia, paleness, neurological disorders, muscle weakness (18, 19)
Manganese (Mn) 0.400–6.400 0.260–0.860 Bone demineralization, skin rashes, hair depigmentation, decreased (19, 20)
serum cholesterol, and increased alkaline phosphatase activity,
increased premenstrual pain in women
Iodine (I) 0.010–0.019 0.0003–0.0065 Goiter (1, 21)
Selenium (Se) 0.001–0.013 0.000095–0.000225 Infertility, myodegenerative diseases, cardiovascular disease, and (16, 22)
cognitive decline
Molybdenum (Mo) 0.0055–0.0449 0.0290–0.130 Encephalopathy (17, 19, 23)
Cobalt (Co) 0.001–0.00225 ND Numbness, tingling in your hands and feet, and severe tiredness (24)
(fatigue)
Nickle (Ni) 0.120–2.530 0.020–0.350 Anemia and parakeratosis-like damage (25)
Vitamin A (Retinol) 5.630–119.460 0.00462–0.818 Impaired immunity, rashes, and ocular effects, i.e., night blindness and (1, 19)
xerophthalmia
Vitamin D (Calciferol) 0.030–0.460 ND Osteomalacia resulting in bone pain, muscular weakness, and weak (1)
bones
Vitamin E (α-Tocopherol) 0.300–11.030 0.00027–0.00443 Hemolytic anemia in infants (16, 19)
Vitamin K (Phylloquinone) 0.0001–0.002 0.130–0.300 Poor bone development, bleeding, increased cardiovascular diseases, (26, 27)
osteoporosis
Vitamin C (Ascorbic acid) 140–9140 ND Scurvy (1)
Vitamin B1 (Thiamin) 0.120–0.900 0.020–0.117 Beriberi or thiamine deficiency (16, 17, 19)
Vitamin B2 (Riboflavin) 0.010–0.220 0.016–0.220 Skin disorders, i.e., angular stomatitis, hair loss, cheilosis, sour throat (16, 17, 19)
Vitamin B3 (Niacin) 1.700–8.200 0.526–1.060 Dementia, diarrhea, could often lead to death (16, 17)
Vitamin B5 (Pentothenic acid) 0.050–0.950 0.0600–0.318 Insomnia, depression, irritability, vomiting, stomach pains, burning feet (17, 19, 27)
Vitamin B6 (Pyridoxine) 0.00015–0.00050 0.039–0.134 Electroencephalographic abnormalities, swollen tongue, depression (16, 19)
and confusion
Vitamin B7 (Biotin) 0.0001–0.002 0.012–0.110 Alopecia and scaly erythematous dermatitis, aciduria, acidemia, (27, 28)
hearing and vision problems, and reduce the growth
Vitamin B9 (Folic acid, Folacin) 0.003–0.078 0.0036–0.067 Megaloblastic anemia having larger red blood cells than usual (16, 19)
Vitamin B12 (Cobalamin) 0.020–0.450 0.0007–0.008 Anemia followed by damaged nerves, can affect thinking and memory (1, 29)
Macronutrients (g/100 g of food)
Carbohydrates 42.000–46.100 20.130–27.420 Siabetic ketoacidosis, hyperosmolar coma, and hypoglycemia (16, 30)
ultimately affecting the central nervous system
Proteins 7.900–9.400 7.550–9.020 Kwashiorkor, marasmus (16, 30)
Fats 39.300–42.700 0.400–2.660 Scaly dermatitis, alopecia, thrombocytopenia, intellectual disability (16, 30)
Potassium (K) 0.150–0.410 0.291–0.391 High blood pressure, muscle weakness, constipation, fatigue (16, 17)
Calcium (Ca) 0.010–0.140 0.019–0.050 Muscles aches, spasms of muscles in the throat, tingling in different (16, 17)
body parts, abnormal heart rhythms
Magnesium (Mg) 0.020–0.120 0.036–0.057 Diabetes, coronary heart disease, hypertension, osteoporosis (16, 17)
Sulfur (S) 0.08–0.160 ND Obesity, Alzheimer’s disease, chronic fatigue (31)
Phosphorous (P) 0.289–0.1160.2 0.114–0.180 Anorexia, ataxia, confusion, anemia, proximal muscle weakness, (1, 17)
skeletal effects, increased infection risk, paresthesias, confusion
Sodium (Na) 0.001–0.0210 0.230–0.930 Brain swallowing is indicated by headaches, seizures, coma, and even (16, 32)
death
Histidine (HI) 2.000–3.800 12.000–19.370 Anemia and low blood levels (16, 30)
Isoleucine (IIe) 3.000–4.200 20.000–30.620 Wasting and weakening of muscles and tremors (16, 30)
Leucine 6.300–14.200 41.900–60.600 Poor growth, weight loss, rashes, hair loss, desquamation, poor (16, 30)
feeding
Lysine (Lys) 2.100–4.300 41.250–48.200 Red eyes, irritability, hair loss, anorexia, nausea, inhibited growth, (16, 30)
fatigue
(Continued)

TABLE 1 | Continued
Nutrient Composition in Quantity in pulses Deficiency symptoms in humans References

cereals
Tryptophan (Trp) 1.000–2.500 1.875–6.900 Anxiety and irritability, aggressions, increased pain sensitivity (16, 30)
Methionine (Met) 1.200–2.500 7.500–18.200 Tremor, low intelligence, abnormal muscle contractions, severe (16, 30)
headache, abnormal eye developments
Phenylalanine (Phy) 4.300–5.500 20.630–50.630 Behavioral problems, small head, seizures (16, 30)
Threonine (Thr) 2.400–4.000 19.400–32.500 Lameness, neurological disorders (16, 30)
Valine (Val) 3.600–6.700 23.200–32.500 Dehydration, hypotonia, loss of appetite, vomiting (16, 30)
Linoleic acid 0.700–3.520 16.000–57.000 Poor growth, fatty liver, reproductive failures, skin lesions (16, 30)
Linolenic acid 0.070–0.190 2.000–46.000 Intellectual disability, scaly dermatitis, alopecia, thrombocytopenia (16, 30)
world’s population uses white rice as a staple food due to its components, and quality protein (44). There is a need to
palatability, taste, and softness. However, its nutritional value enhance tryptophan and lysine content to increase nutritional
is very low, it has a low concentration of vitamin E (0.0075– quality of vital zein proteins. The opaque-2 (o2) mutant form
0.30 mg/100 g), niacin (1.3–2.4 mg/100 g), riboflavin (0.02–0.06 in maize has the potential to increase tryptophan and lysine
mg/100 g), thiamine (0.02–0.11 mg/100 g), fiber (0.2–0.5 g/100 g), contents. Varieties possessing o2 allele have relatively high lysine
fats (0.2–0.5 g/100 g), protein (6.3–7.1 g/100 g), and traces of and tryptophan contents as compared to wild typed varieties
Zn (0.3–2.1 mg/100 g) and Fe (0.2–2.7 mg/100 g). While brown (45, 46). Micronutrients, i.e., zinc, iron, health-supporting γ-
rice has enough vitamin E (0.9–2.5 mg/100 g), niacin (3.5– linolenic acid, polyunsaturated fatty acids, and stearidonic acid
5.3 mg/100 g), riboflavin (0.04–0.14 mg/100 g), thiamine (0.29– (STA), are also reportedly deficient in barley. Barley seeds have
0.61 mg/100 g), fiber (0.6–1.0 g/100 g), fats (1.6–2.8 g/100 g), a phytase gene (HvPAPhy a) that is responsible for enhanced
protein (7.1–8.3 g/100 g), and relatively high amounts of Zn (1.5– phytase action. This increased phytase activity improves zinc and
2.2 mg/100 g), and Fe (0.7–5.4 mg/100 g), which are essential iron bioavailability. Due to overexpression (Box 1) of cellulose
for proper growth and development. Hence, it is strongly synthase-like gene (HvClF), β glucans content increases. These
recommended to use brown rice to harvest maximum dietary are the dietary fibers involved mainly in the reduction of type II
benefits (41). Iron (Fe) is considered the most important element diabetes and cardiovascular disease (6). Sorghum is also reported
for health, with its deficiency leading to severe anemia. Similarly, to be deficient in iron and zinc along with iodine, selenium,
deficiency of β-carotene and niacin leads to childhood blindness magnesium, and calcium (47).
and pellagra (in which a person has wounds in the mouth),
dementia, and diarrhea, respectively (9).
Pulses
About 1,000 pulses are known to humans but only 20 of
WHAT TO BIOFORTIFY? them are cultivated for human consumption including cowpea
(Vigna unguiculata), pigeon pea (Cajanus cajan L.), chickpea
Biofortification ensures maximum uptake of minerals, their (Cicer arietinum L.), urban (Vigna mungo L.), mung bean
transportation to eatable parts, and bioavailability. There is (Vigna radiata L.), French bean (Phaseolus vulgaris), lentil
an utmost need to biofortify cereals and pulses for essential (Lens culinaris Medik), horse gram (Macrotyloma uniflorum),
micronutrients for the provision of balanced diets to the masses soybeans (Glycine max), moth bean (Vigna aconitifolia), Lathyrus
(5, 42). The following section briefly describes deficiencies of (Lathyrus sativus L.), field pea (Pisum sativum L.), etc. (5).
different cereals and pulses crops that need to be biofortified. Pulses are deficient in iron and zinc contents like the majority
of cereals (42). These are also deficient in selenium, dietary
Cereals fibers, and sulfur-containing amino acids including cysteine
The commonly cultivated cereal crops include rice, wheat, maize, and methionine (48). Although pulses are rich in proteins and
sorghum, barley, millets, and oats. Rice is reportedly deficient micronutrients, there are some anti-nutrients as well which
in Fe, Zn, and pro-vitamin A, which is a leading cause of restricts the bioavailability of proteins and nutrients, e.g., lectins,
hidden hunger in rice-eating areas around the globe and causes phytic acid, saponins, lathyrogens, protease inhibitors, α-amylase
anemia, night blindness, and loss of vision (9). Wheat needs inhibitors, and tannins, restrict the absorption of iron, zinc,
biofortification for effective provision of zinc, iron, selenium, calcium, magnesium, and other essential nutrients (49). There
grain yellow pigment contents (GYPC), pro-vitamin A, grain is a need to understand the metabolic pathway involved in the
anthocyanins, and essential amino acids, similarly some anti- synthesis of these compounds and the disruption of key genes
nutrient compounds, i.e., phytic acid, are present in excessive involved for their reduced production.
quantities which needs to be checked (43). Maize is found As we are talking about the removal of anti-nutrient
deficient in vitamin E (tocopherol, tocotrienol) along with compounds from cereals and pulses, we must also consider
persuasive antioxidants, vitamin C, vitamin A, anti-nutrient their vital functions in plant development as discussed below.

Lectins: are a diverse family of plant proteins found in almost all promise for biofortification by soil and foliar application. The
organisms including animals, microorganisms, and plants, which most used fertilizer is nitrogen, phosphorus, and potassium
restricts the bioavailability of iron and zinc. There are over 500 (NPK) based, which are essential for both plant and human
members of lectins in plants that are produced in response to health. Some other micro-minerals like iodine, copper, zinc, iron,
biotic stresses, i.e., disease, insect, molds, and fungi attack and nickel, manganese, molybdenum, etc. are also essential for crops.
serve as the first defense line (50). Oxalates: or oxalic acid, are Most of these micronutrients are readily available in the soil
substances that can form insoluble salts with sodium, potassium, and absorbed by plants and become part of the food chain.
magnesium, iron, and calcium and restricts their bioavailability. But sometimes plants are unable to absorb them and hence are
These are produced in all photosynthetic organisms for calcium applied in the form of mineral nutrition (62).
regulation, detoxification of heavy metals, and plant protection Apart from agronomic biofortification (Box 1), conventional
(51). Phytate: phytic acid or myo-inositol hexaphosphate (IP6) breeding (Box 1) relies on the genetic variability for the trait
is another plant-derived anti-nutrient compound that restricts of interest (TOI) in the gene pool (55). The desired genes
the bioavailability of iron and zinc. It usually serves as a storage are pyramided using the conventional crossing techniques
house for plant phosphates, antioxidants for germinating seeds, followed by extensive screening of segregation populations.
and energy sources. These are usually produced during seed The Wheat Research Institute (WRI), Faisalabad and National
germination and serve as a source of 50–60% phosphorus during Agriculture Research Council (NARC), Pakistan had released
germination (52). Tannins: are a class of high molecular weight biofortified wheat varieties, i.e., “Zincol” and “Akbar-2019” back
polyphenols that are found in the plant kingdom and serve as in 2015 and 2019, respectively, with enhanced iron and zinc
anti-nutrients by chelating iron, zinc, and copper. There are contents (63). Similarly, Bangladesh Rice Research Institute
two classes of tannins, i.e., hydrolyzable tannins and condensed (BRRI) released high iron and zinc rice varieties, i.e., Dhan
tannins. Condensed tannins are found abundantly in plants and 62, 64, 72, and Jalmagna (9). Many common bean varieties
play a role in plant defense as an antioxidant, anti-carcinogenic, including MAC42, CAB2, RWV2887, 1129, 3317, 3006, and
immunomodulatory, detoxifying, and cardio-protective activities 33166, MAC44, RWR2154, PVA1438, HM21-7, CODMLB 3,2
(53). Given the important metabolic activities of anti-nutrient and 001, RWR2245, and Cuarentino with high zinc and iron
compounds, there is a need to tradeoff between their desirable contents were released to reduce malnutrition. Some lentils
and undesirable functions before their silencing or knocking verities Idlib3, Idlib2, Alemaya, L4704, Shital, Simal, Sisir,
out (54). Khajurah2, ILL 7723, Barimasur 8, 4, 7, and 5 with high zinc
contents were developed and released (55, 64). Further varieties
developed through conventional breeding are summarized in
STRATEGIES TO ADDRESS Supplementary Table 1. Crop wild relatives (CWRs) harbor
MALNUTRITION important genes that are the source of essential micronutrients.
To achieve standard nutritional concentrations and protein
Many direct and indirect approaches are used to increase requirements, the introgression of genetic variation from crop
concentration of essential nutrients in grain and other parts wild relatives to modern crops is inevitable (65). The most
of plants mainly to overcome malnutrition (55). Dietary efficacious example of wild hybridization is the introgression
diversification (Box 1), artificial supplementation (Box 1), and of Gpc-B1 locus in bread wheat (Triticum aestivum) from wild
biofortification are used to overcome malnutrition. However, emmer (Triticum turgidum ssp. dicoccoides) via chromosomal
biofortification is cost-effective, feasible, and a long-term solution substitution. This introgression was highly fruitful because it
to reduce this nutrient deficiency. The biofortified crops have all delivered wheat plants with an improved nutritional profile (Mn,
the essential vitamins, minerals, and fatty acids that are required Fe, and Zn) without yield penalty (66).
by a human to fulfill his or her nutritional demands (56–58).
Furthermore, biofortified crops (Box 1) are eco-friendly as these HarvestPlus Program
enhance nutrient uptake from soil and improve soil health and It is worthwhile to mention the contribution of the HarvestPlus
these are sustainable as well (59, 60). For biofortification different program, which is working in connection with agriculture and
agronomic, conventional breeding, transgenic, and gene editing nutrition to end micronutrient deficiencies and hidden hunger
approaches are used as described below. globally. It is targeting the most vulnerable communities and
providing them a food-based solution to control micronutrient
Conventional Approaches deficiencies. The organization is working alongside its partners
Biofortification through agronomic approaches is an economical to develop crop varieties that are rich in iron, zinc, and vitamin
and easy method but the method of nutrient application, A, the three nutrients that are most lacking in global foods as
their type, and environmental factors requires great care. This identified by World Health Organization (WHO) (67).
approach focuses on enhanced nutrient availability to the plants, It is working for the development of nutritionally enriched
their effective utilization, and mobility in plants, and an increase crops that could be evidence-based, cost-effective, and
in microbial activity for their efficient utilization (Figure 1). sustainable using conventional breeding approaches. The
Microbes like rhizobium, bacillus, azotobecter, actinomycete, and program is aimed at reaching 1 billion individuals with a supply
some fungal strains, i.e., P. indica, are used to enhance nutrient of biofortified crops by 2030. HarvestPlus is currently focused
availability and uptake (61). Mineral nutrients also hold good in Pakistan, India, Bangladesh, the Democratic Republic of

FIGURE 1 | Different methods for biofortification of crops. These are broadly classified into three types, i.e., agronomic techniques, biofortification of crops through
foliar spray of micronutrients. Foliar application helps in acquisition of more nutrients in reproductive parts, and hence healthier foods are delivered to the consumer. In
this technique, nutrients are applied in liquid form in aerial parts of plants and got absorbed through stomata and epidermis and become part of the food chain.
Mineral fertilization through flooding for biofortification of crops. Minerals, i.e., selenium, zinc, calcium, etc., are supplied to crops alongside irrigation and are readily
available for uptake and as a result their accumulation in eatable parts of plants is increased. Mineral fertilization through soil application for biofortification of crops.
Mineral fertilizers, usually NPK, are applied in the soil bed before sowing or alongside seed using different seed cum fertilizer drills and as a result they are absorbed
and made part of the food chain through root uptake. Microbe-mediated enhanced uptake of nutrients for biofortification of crops. Different microbial species, i.e.,
rhizobium bacteria, mycorrhizae fungi, etc., help plants in nutrient acquisition through mutualism. Conventional breeding helps in biofortification of crops by crossing
two parents possessing contrasting phenotypes and selection in subsequent segregation generations based on trait of interest. New breeding techniques, knocking
out of genes involved in biosynthesis of anti-nutrient compounds. Different anti-nutrient compounds, i.e., lectins, phytic acid, saponins, lathyrogens, protease inhibitor,
α-amylase inhibitors, and tannins restrict bioavailability of essential micronutrients. Hence, this results in malnutriated crops. Genes involved in biosynthesis of
anti-nutrients could be repressed through RNAi for reduced accumulation of these compounds. Overexpression of gene responsible for micronutrient accumulation in
plants leads toward micronutrient biofortification. Overexpression of genes leads toward increased micronutrients accumulation and, as a result, more deposition in
the eatable plant parts. Biofortification of crops through gene transfer from other species has resulted in improvement of nutritional quality of crops and alleviation of
(Continued)

FIGURE 1 | malnutrition. Different genes involved in biosynthesis of pro-vitamin A (CrtB), iron homeostasis (Fer1-A), and flavonoids production (C1) has been
transferred across species for biofortification. These genes help in the production of a balanced diet. As a result, different diseases associated with malnutrition are
controlled, e.g., Golden Rice has helped to overcome night blindness and other diseases associated with pro-vitamin A deficiency. However, there are certain
limitations of this technology, i.e., laborious, expensive, time consuming, and above all else, it has less public acceptability due to regulatory issues.
TABLE 2 | Summary of ongoing activities and achievement of HarvestPlus program.
Country Crop Nutrient Varieties developed
Pakistan Wheat Zinc Zincol-2015, Akbar-2019

Bangladesh Rice Zinc BRRI dhan62, BRRI dhan64, BRRI dhan72, BU Aromatic Zinc Rice
Democratic Republic of Congo Cassava Vitamin A Kindisa (TMS 2001/1661), Lumonu, Vimpi
Maize Vitamin A PVA-SYN12F2 (composite), SAM 4 VITA (composite), GV605 (hybrid), GV604 (hybrid)
Beans Iron HM 21-7 (bush), COD MLB 032 (bush), RWR 2154, RWR 2245 (bush), COD MLV
059 (bush), PVA 1438 (bush), Nain de Kyondo (climber), Namulenga (climber),
Cuarentino (climber)
India Pearl millet Iron ICMH 1201 (Shakti-1201), ICTP 8203-Fe-10-2 (Dhanashakti)
Wheat Zinc BHU-6 (Chitra) and BHU-3
Latin America and the Caribbean Cassava Vitamin A No data
Beans Iron No data
Maize Zinc No data
Rice Zinc No data
Sweet potato Vitamin A No data
Nigeria Cassava Vitamin A TMS 01/1368—UMUCASS 36, TMS 07/0593—UMUCASS 45, NR
07/0220—UMUCASS 44, TMS 07/539—UMUCASS 46, TMS 01/1371—UMUCASS
38, TMS 01/1412—UMUCASS 37
Maize Vitamin A Sammaz 37, 38, and 39, Ife Hybrid 4, Oba Super 6, SC 510, SDM4, Orange Maize,
PVA 2 (Kapam 6)
Rwanda Beans Iron RWR 2245 (bush), MAC 44 (climber), RWV 1129 (climber), RWR 2154 (bush), CAB 2
(climber), RWV 3317 (climber), MAC 42 (climber), RWV 3006 (climber), RWV 3316
(climber), RWV 2887 (climber)
Uganda Bean Iron Roba 1 (bush)
Sweet potato Vitamin A Ejumula, Vita, Kakamega, Naspot 12 O, Naspot 13 O, Kabode
Zambia Maize Vitamin A GV662A (Kamano Seed), GV665A (SeedCo), GV664A, GV671A (ZamSeed), GV573
(Advanta), GV672A (Afriseed)
Sweet potato Vitamin A Olympia, Chiwoko, Twatasha, Zambezi, Kokota, Mansa Red, Chingovwa
Zimbabwe Maize Vitamin A ZS242, ZS246, ZS244, ZS248
Beans Iron No data
Note the information was obtained from the official website of HarvestPlus (https://www.harvestplus.org/).
Congo, Latin America and the Caribbean, Nigeria, Rwanda, Overall a total of 242 crop varieties has been released in
Uganda, Zambia, and Zimbabwe. The program was divided 30 countries since the inception of HarvestPlus, 8.5 million
into three phases, i.e., discovery phase (2003–2008), which smallholder farming households are growing these varieties and
was aimed at the identification of high-risk populations, their distributing them to 42.4 million people around the globe. The
food consuming habits, plant breeding resources, feasibility program was focused on the development of zinc biofortified
studies, and a pilot project to deliver pro-vitamin A enriched wheat in Pakistan and rice in Bangladesh, vitamin A enriched
sweet potato in Africa. The development phase (2009–2013) cassava, maize, and iron enriched beans in the Democratic
was aimed at the evolution of biofortified crop varieties about Republic of Congo, and iron and zinc enriched peal millet and
iron, zinc, and pro-vitamin A in target countries, their testing at wheat in India (https://www.harvestplus.org/where-we-work).
multiple locations for stability analysis, and researchers assessed The details of other countries along with success stories in the
the nutritional profile of developed varieties and country-wise form of biofortified varieties evolved are summarized in Table 2.
teams were developed for delivery of biofortified crops. The
delivery phase (2014-onward) was focused on the creation of New Breeding Techniques
consumer demands for biofortified crops to reach maximum New breeding techniques (Box 1), i.e., transgenic breeding
populations. Researchers were working in the estimation of the (Box 1), RNA interference (RNAi) (Box 1), and genome editing
area covered by biofortified crops and making efforts for its (Box 1), are playing key roles in the biofortification of crops by
long-term sustainability (68). opening new avenues for the creation of novel genetic variation

TABLE 3 | Summary of studies explaining biofortification of crops through transgenic breeding and genome editing.
Technique Crop Micronutrient Genes involved References
Transgenic Triticum aestivum Iron TaFer1-A↑, OsNAS2↑, TaVIT2↑, GmFER↑, PhyA↑, NAM-B1↑, (12, 78)
Tyrosine, lysin, Ama1↑ (79)
cysteine, methionine
Vitamin A CrtI↑, CrtB↑, Bacterial PSY↑ (77)
Zinc OsNAS2↑, NAM-B1↑, PhyA↑ (12, 78)
Oryza sativa Zinc IDS3↑, HvNAAT-A↑, HvNAAT-B↑, HvNAS1↑, OsNAS3↑, OsNAS1↑, (12, 80, 81)
OsNAS2↑, AtNAS1↑, AtIRT1↑, Pvferritine↑, Afphytase↑, OsFer2↑,
OsHMA1↑, OsYSL15↑, OsYSL2↑, OSIRO2↑, OsVIT1↓, OsVIT21↓,
OsYSL9↓
Iron AtNAS1↑, AtIRT1↑, Pvferritine↑, Afphytase↑, OsFer2↑, OsIRT1↑, (12, 80, 81)
OsNAS1↑, OsNAS2↑, GmFER↑, OsVIT1↓, OsVIT2↓, HvNAS1↑,
OsYSL2↑, OsIDEF1↑, OsNAC5↑, OsYSL9↓
Vitamin E AtTC↑, AtHP↑ (82)
Vitamin B1 THIC↑, THI1↑, TH1↑ (83)
Vitamin B6 AtPDX1.1↑, AtPDX02↑ (84)
Vitamin B9 ADCS↑, AtGTP cyclohydrolase 1↑ (85)
Provitamin A Carotene desaturase↑, daffodil PSY↑ (80)
Flavonoid, linoleic acid GmFAD3↑, ZmC1↑, chalcone synthase↑, phenylalanine ammonia (86)
lyase↑
Zea mays Iron GmFER↑, aspergillus phytase↑, aspergillus phy2↑ As proposed by
(55)
Lysine Sb401↑ (77)
β-carotene Zmpsy 1↑ (87)
Vitamin A crtI↑, crtB↑ (77)
Tocopherol, tocotrienol HGGT↑ (87)
Sorghum bicolor Pro-vitamin A PSY-1↑, CRT-I↑, PMI↑, LPA-1↑↑ (88)
Hordeum vulgare Zinc Zn transporter gene↑, DHPS↑ As proposed by
(55)
Iron Phytase↑, AtZIP1↑, (12, 89)
Lysine DHPS (89)
Vitamin E AtVTE3↑, AtVTE4↑ (90)
β-glucans HvCs1F↑ (90)
Cicer arietinum Iron GmFER↑, NAS2↑ (91)
Phaseolus vulgaris Methionine Methionine rich storage albumin↑ (92)
Glycine max Provitamin A Carotene desaturase↑, crtB↑, crtW↑, bacterial PSY↑, bkt1↑ (93)
Fe and zinc Phytase↑ (12)
Lysine Aspaktokinase↑, dihydrodipicolinic acid↑ (94)
Cysteine Maize zein protein↑, O-acetyl serine sulfhydrylase↑ (94)
Methionine Cystathionin γ-synthase↑, maize zein protein↑ (95–97)
Genome editing Rice Fe OsVIT2↑ (98)
Amylose contents Wx gene↓ SBEI↓, SBEII↓, OsWaxy↓ (99, 100)
β-carotene OsCCD4a↑, OsCCD4b↑, OsCYP97A4↑, OsCCD7↑ and OsDSM2↑, (12, 101)
Osor↑
2 AP production and Badh2↑ (100)
fragrance
Protein contents OsNRT1.1B↑ (102)
Phytic acid OsITPK1-6↓ (103)
Thiamine Ostpk1↑, Ostpk2↑, Ostpk3↑, Osncs1↑, OsThiC↑, As proposed by
(104)
Wheat Fe and Zn TaVIT2↑ (105)
Grain weight TaGW2↑ (106)
Low gluten Alpha-gliadin 33-mer↓ (107)
Maize Branched amino acids ZmALS1↑ and ZmALS2↑ (108)
synthesis
Carotenoid synthesis ZmPSY1↑ (109)
Note:↓ indicates the downregulation/knockout of gene, whereas ↑ indicates upregulation/overexpression of the targeted gene.

that does not exist in the gene pool as reviewed in Van Der µg Fe/g DW, Osfer2 to 7.0 µg/g DW, while OsNAS2 increased it
Straeten et al. (69). to 19 µg/g DW (114). Moreover, overexpression of ferritin under
control of endosperm-specific promoters’ globulinb1 (OsGlb1 )
Transgenic Breeding and glutelin B1 (OsGluB1 ), NAS with control of OsActin1
Cereals promoter and OsYSL2 along with OsGlb1 and OsSUT1 transport
Biofortification through transgenic breeding is an efficient, promoters resulted in iron biofortification in polished rice
sustainable, and cost-effective approach to combat malnutrition. (115). Similarly, zinc biofortification with HvIDS3, HvNAAT-
Transgenic crops (Box 1) are constituted when limited genetic A, HvNAAT-B, and HvNAS1 genes resulted in 35 µg Zn/g of
variability is present in the gene pool and wild relatives. DW (58), soybean ferritin, Aspergillus flavus phytase, OsNAS1
Transgenic breeding helps in biofortification through the exhibited 35 µg Zn/g of DW, while overexpression of OsNAS2
introduction of genes that either decrease anti-nutrient resulted in 76 µg Zn/g of DW in rice crop (11). Another study
compounds or increase micronutrients accumulation and highlighted the biofortification of endogenous rice lysine-rich
bioavailability (Figure 1) (12). Detailed discussion about histone proteins by overexpression of RLRH1 and RLRH2 with
transgenic cereals and pulses developed for micro- and modified rice glutelin1 promoter. As a result lysine contents
macronutrients (like vitamins, fatty acids, minerals, and amino increased up to 35% in transgenic rice in comparison to wild
acids) biofortification is summarized below. type (116).
The deficiencies of micronutrients like zinc, iron, vitamin Similarly, overexpression of rice genes OsNAS1, OsNAS3,
A, and proteins in cereal grains could be overcome by the OsIRTI, and OsNAS2 accumulated double the amount of zinc.
introduction and overexpression of genes corresponding to In rice AtNAS1, AtIRT1, Afphytase, and Pvferritin genes were
these micronutrients. For example, high iron accumulation in also introduced to increase iron and zinc concentration and
wheat grain was obtained through ectopic overexpression of results were found satisfactory in polished rice. The disruption
TaFer1-A and OsNAS2 genes which increased iron content of activity of vacuolar iron transporter OsVIT2 and OsVIT1 gene
up to 80 and 85 µg/g in grains, respectively, but TaFer1- also increased iron and zinc concentrations in rice grain (58).
A expression is not uniform across generations (71, 72). The overexpression of amino deoxy-chorismate synthase ADCS
Similarly, overexpression of TaVIT doubled the iron contents and AtGTP-cyclohydrolase1 genes has enhanced folic acid content
in wheat grain; however, distribution was not uniform for in rice (117). Golden Rice has high pro-vitamin A accumulation
whole grain (73). Overexpression of the Amaranthus albumin- and it was considered the best example of biofortification using
based Ama1 gene overcomes essential proteins and the amino transgenic crops (118). The antioxidant activity in rice is linked
acid deficiency (tyrosine, lysine, cysteine, and methionine) (74). with flavonoids that can be enhanced through overexpression
Overexpression of the phytochrome (PhyA) gene enhanced of chalcone synthase (CHS), maize C1, phenylalanine ammonia
phytase production and anti-nutrient activity, whereas silencing lyase, and GmFAD3 genes resulting in increased accumulation of
of phyA and TaABC13 transporter gene decreased phytic acid flavonoids and linoleic acid accumulation in rice (119).
production (18–19%) and increased iron and zinc bioavailability In maize endosperm, vitamin A accretion was increased by
(75). The silencing of the SBEIIA gene increased concentration overexpression of bacterial crtI and crtB gene under the control
of less digestible amylose starch to combat obesity and over- of endosperm specific super gema zein promotor to as high as
nutrition (Box 1). The overexpression of carotene desaturase 9.8 µg/g of DW. Similarly, overexpression of maize Psy1 gene
CrtI, CrtB, and PSY gene from bacteria also increased vitamin also increased β-carotene accumulation in maize. Overexpression
A concentration (73). A higher concentration of vitamin A, of the dehydroascorbate reductase gene increased vitamin C
4.96 µg/g DW, was attained after using maize PSY1 gene accumulation in maize by changing ascorbic acid oxidizing form
encoding phytoene synthase and bacterial Crtl genes (76). CrtB into the reducing form (120). The overexpression of aspergillus
or Crtl also resulted in increased accumulation of pro-vitamin A phytase, aspergillus niger phy2, and soybean ferrite gene in maize
to as high as 3.21 µg/g of DW (77). enhanced iron bioavailability. The overexpression of the potato
Among cereal crops, rice is biofortified with vitamin A, B1, sb401 gene enhanced lysine contents in maize grain. Antisense
B6, E iron, and zinc and other compounds (Table 3). Researchers dsRNA targeting α-zeins enhanced tryptophan and lysine
indicated that vitamin A biofortification was done via Phytoene accretion in maize. The overexpression of the Homogentisic acid
synthase (PSY) extracted from daffodil while phytoene desaturase Geranyl Geranyl Transferase (HGGT) gene enhanced tocopherol
(Crtl) genes extracted from Erwinia uredovora were later and tocotrienol concentrations in maize (55) and resultantly
incorporated in Agrobacterium tumefaciena, which increased biofortified maize was obtained. Furthermore, overexpression of
vitamin A concentration to 1.6 µg/g of DW (110). Another PSY1 maize also confirmed a higher concentration of vitamin A
research highlighted that the PSY gene extracted from maize 59.32 µg/g of DW (121, 122).
and Crtl from Erwinia uredovora also enhanced vitamin A Sorghum transgenic line homo188 harbors different genes,
concentration to 37 µg/g DW. Moreover, PSY and lycopene β- i.e., PSY-1, CRT-I, PMI, LPA-1, for enhanced pro-vitamin
cyclase (β-lcy) extracted from daffodil also exhibited an increase A content and has high nutritional value (88). Through
in vitamin A concentration of around 1.6 µg/g DW (111–113) in overexpression of zinc transporter genes in barley, the amount
rice. An overexpression of soybean ferritin gene Soyfer H-1 gene of zinc was enhanced. The expression of DHPS and phytase gene
resulted in an increase of Fe contents to as high as 38.1 µg Fe/g causes an increase in lysine iron and zinc bioavailability in barley.
of DW, Phaseolus ferritin improving iron concentration to 22.07 The co-expression of the 2-methyl-6-phytyl benzoquinol methyl

transferase AtVTE-4 and AtVTE3 gene reportedly enhanced most transgenes are not discussed in detail and are summarized
vitamin E in barley seeds. Likewise, the expression of the D6D in Table 3.
gene enhanced stearidonic acid and linolenic acid content in
barley, which are essential for human health. The concentration
of dietary fibers like β-glucans was also escalated barely through RNA Interference
the overexpression of the cellulose synthase-like (HvCslF) gene RNAi is a sequence-specific gene regulation process driven by
(6). Transgenic millet reportedly exhibited higher zinc content a double-stranded RNA (dsRNA) molecule, which results in
owing to zinc transporters, while transcriptomics indicated inhibition of either transcription or translation of a particular
higher calcium sensor genes involved in uptake, translocation, gene. Since its discovery, RNAi has opened a new vista for crop
and accumulation of calcium in finger millet. Moreover, anti- improvement. It is a precise, stable, efficient, and better tool
nutrients including phytic acid, polyphenols, and tannins are also than antisense technology. RNAi provides a platform for the
reported to have a lower concentration in the transgenic millet incorporation of biotic and abiotic stress tolerance and delivery
arisen through genome editing (123). of quality food through biofortification and bio-elimination. It
is widely used for nutritional quality enhancement of crops and
Pulses removal of food allergens and contaminants (126, 127). The
Transgenic breeding was less explored in pulses for following sections cover some examples of the use of RNAi for
biofortification and few pulses like chickpea, common bean, the biofortification of cereals and pulses.
soybean, and lupines are modified to overcome malnutrition Phytic acid (PA) is considered a major anti-nutrient in cereals
(124). The basic aim of biofortification of pulses through and pulses due to its ability to chelate micronutrients and restrict
transgenic breeding is the enrichment of essential amino acids, the bioavailability of important nutrients. RNAi was employed
iron and zinc fortification, and reduction of anti-nutrients to disrupt an important gene [inositol pentakisphosphate kinase
compounds. The deficiency of sulfur-rich amino acids was (TaIPK1)] of the PA biosynthesis pathway in wheat. The resultant
overcome through overexpression of heterologous proteins rich homozygous transgenic lines of wheat at the T4 stage show
in these amino acids. Maize-based 27 kDaγ-zein, a cysteine-rich reduced expression of TaIPK1, reduced PA accumulation (26–
protein, was introduced and overexpressed in different pulses 58%), and increased grain phosphate, zinc, and iron contents
crops for nutritional enhancement of cysteine amino acid (128). Carotenoid contents have high nutritional value being
(48). Similarly, methionine concentration was enhanced in precursors of pro-vitamin A. In rice, carotenoid contents
narbon bean and lupin by overexpression of S-rich proteins. were enhanced by RNAi-based disruption of carotenoid-
The concentration of methionine was enhanced in Brazil cleavage dioxygenases (CCDs) genes which degrade carotenoids.
nut by overexpression of the 2S albumin storage protein and Three genes, i.e., OsCCD1, OsCCD4a, and OsCCD4b, were
aspartate kinase using seed-specific promotor and 4 times more disrupted using RNAi. Resultantly an increase in carotenoid
methionine accumulation was achieved in seed (48). The rice production was observed in mutants, i.e., OsCCD1-RNAi (1.4-
OASA1D transgene enhanced free tryptophan accumulation in fold), OsCCD4a-RNAi (1.6-fold), and OsCCD4b-RNAi (1.3-fold)
adzuki bean upon transformation (48). as compared to wild types (129). Lysine is the main limiting
Glycine max ferritin and chickpea NAS2 genes were essential amino acid in rice, which serves as a source of energy
introduced and overexpressed to increase iron bioavailability in and nutrition. Two enzymes, i.e., aspartate kinase (AK) and
chickpea (91). In soybean overexpression of cystathionine dihydrodipicolinate synthase (DHDPS), are liming factors for
γ-synthase gene enhanced methionine concentration. lysine and are extremely sensitive to a feedback mechanism.
Overexpression of maize zein protein in soybean enhanced RNAi-based repression of AK and DHDPS enzymes was carried
cysteine and methionine content in seeds. The overexpression out in rice and resultantly 6.6- and 21.7-fold more lysine was
of the O acetyl serine sulfhydrylase gene also enhanced accumulated in mutant lines, respectively. When both mutations
cysteine content in seeds. In transgenic soybean upregulation were combined in one genotype, the lysine level was 58.5-fold
of dihydrodipicolinic acid synthase and aspartokinase gene more in wild types (130). Maize endosperm is deficient in pro-
enhanced lysine content in seeds (55). Overexpression of vitamin A and β-carotene leading to vitamin A deficiency in
carotene desaturase and bacterial PSY, bkt1, crtW, and crtB masses. However, other carotenoids, i.e., zeaxanthin which is
gene enhanced pro-vitamin A accretion in soybean (125). produced from β-carotene via a two-step hydroxylation reaction,
The silencing of the ω-3 FAD3 gene was accomplished using are found in sufficient quantity which may be stopped to
siRNA-mediated knockout to reduce α-linolenic concentration increase β-carotene content in maize endosperm. Considering
in soybean. Similarly, the 16-desaturase gene was overexpressed the abovesaid phenomena two maize genes ZmBCH1 and
to increase α-linolenic conversion to its stable form γ-linolenic ZmBCH2 involved in hydroxylation reaction were knocked out
acid and ω-3 fatty acids. Production of isoflavone in soybean was using RNAi to enhance β-carotene in maize endosperm. Mutants
enhanced by maize C1 and R transcription factors drove gene for ZmBCH2 genes showed a significant increase in β-carotene
activation (3, 55). The methionine contents in common beans contents in maize grain indicating that this gene has a key role in
were enhanced by the transformation of methionine-rich storage conversion of β-carotene to other carotenoids (131). Due to the
albumin from Brazil nuts. Similarly, the S-rich amino acid profile presence of γ-kafirin, sorghum was considered less digestible and
of lupins was improved by the transformation of the respective through suppression of γ-kafirin 1A, γ-kafirin1, 2, and γ-kafirin
gene from sunflower and albumin (55). Due to space limitation, through RNAi silencing its digestibility was improved (55).

FIGURE 2 | CRISPR-Cas system mediated targeted genome editing for biofortification of crops. The process begins by selection of cultivar having anti-nutrient
genes, i.e., MRP1, BADH2, INO2, etc., followed by CRISPR/Cas system mediated modification without disturbing the genetic makeup of the rest of the plant and
(Continued)

FIGURE 2 | resultant mutants are obtained in a short period of time as compared to traditional mutagenesis. The mutant plants obtained through this technique are
used in a breeding program for the development of nutritionally improved and transgene free varieties. The illustration also explains pros and cons of genome editing in
comparison to old breeding techniques, i.e., the efficiency of conventional breeding is very low as compared to genome editing. Similarly, off-target effects of
traditional mutagenesis are very high as compared to genome editing.
Although the role of RNAi in pulses biofortification was of nitrogen (102). CRISPR-Cas9 knock out of OsITPK1-6 leads
not elaborated that much, potential also exists for improvement to low phytic acid accumulation in rice grain and resultantly
of pulses. Phytate restricts the bioavailability of micronutrients increases micronutrient availability (103).
in pulses, studies have reported the complex formation of Zinc concentration was increased in wheat through targeted
phytic acid with calcium, magnesium, copper, and iron, mutagenesis of the TaVIT2 gene via genome editing (137).
thereby reducing their solubility properties (4). Some other Four genes belonging to the Alpha-gliadin gene family in wheat
compounds, i.e., prebiotics mainly inulin and fructans, absorb code for high molecular weight gluten (HMWG) protein.
iron, zinc, and calcium in themselves and restrict phytic acid Genome editing was also exploited for the biofortification
activity. Similarly, β-carotene also promotes the absorption of of maize in different regulatory pathways. Acetolactate
iron and zinc in lentils, peas, and chickpeas. Thereby genes synthase genes (ZmALS1 and ZmALS2) were edited for the
encoding these compounds could be overexpressed for enhanced accelerated synthesis of branched-chain amino acids, i.e., aline,
micronutrient bioavailability (132). Selenium is likewise reported leucine, and isoleucine (108). The phytoene synthase gene
to enhance the bioavailability of iodine in lentils, peas, and (ZmPSY1) involved in carotenoid biosynthesis pathway was
chickpeas. However, some inhibitors preventing bioavailability modified using the CRISPR-Cas9 type II system and stable
need further investigation. The biosynthetic pathways involved transformation was observed in progeny; however, functional
in the production of anti-nutrients should be studied and genes characterization of mutants for carotenoid concentration
that play a key role should be silenced or knocked out using RNAi remained in progress (109).
for the development of nutritionally enriched pulses crops (42).
Pulses
Genome Editing Currently, the literature is deficient in examples explaining
Sequence-specific nucleases (SSNs) are used in plant genome the role of genome editing for the biofortification of pulses.
editing (GE) for stably inherited and targeted gene modification However, the potential exists in the exploitation of genome
in the desired crop to produce transgene-free plants. Various editing for iron and zinc biofortification; carried out by
types of SSNs, i.e., TALENs, ZFNs, and CRISPR-Cas system are manipulation of iron-regulated transporter (IRT), ferric-chelate
used for plant genome editing (133). Genome editing through reductase oxidase (FRO), YELLOW STRIPE 1-like (YSL),
CRISPR involves Cas9/13, RNA-guided DNA endonucleases natural resistance-associated macrophage protein (NRAMP),
guided by a short guided RNA (sgRNA) resulting in a complex zinc-regulated transporters, and iron-regulated transporters like
at the target site for targeted gene editing (Figure 2) (127, protein (ZIP) for an increased iron and zinc uptake in all pulses
134). Genome editing was exploited less for biofortification of crops (124).
cereals and pulses; however, some highlighted examples are Another approach employed for the biofortification of pulses
discussed below. genome editing is to target anti-nutrient genes which are
responsible for the reduced bioavailability of micronutrients.
Cereals Saponins are the anti-nutrient compounds that—in lower
The rice OsVIT2 gene was knocked down for increased Fe concentrations—are beneficial; however, if consumed in higher
availability through genome editing (98). CRISPR-Cas9 was quantities, they can act as anti-nutrients (42). An Arabidopsis
used for targeted insertion of 5.2 kb carotenogenesis cassette thaliana-based study indicated that 13 OSC genes, 246 P450
comprising of Ctrl and PSY genes of maize driven by genes, and 112 uridine diphosphate glycosyltransferases (UGTs)
endosperm specific promotor in the rice line Kitaake. The are involved in the biosynthesis of saponins. There is a need
resultant mutants accumulated 7.9 µg/g dry weights (DW) β- to identify key regulatory genes in the saponins biosynthesis
carotene in endosperm, which is comparable to the Kaybonnet pathway and eliminate them for reduced saponins production
rice variety developed through traditional transgenic breeding (138). Similarly, genes responsible for the production of other
commonly known as Golden Rice2 (135). Five rice carotenoid anti-nutrients, i.e., lathyrogens, protease inhibitor, and α-amylase
catabolic genes (OsCCD4a, OsCCD4b, OsCYP97A4, OsCCD7, need to be identified and consequently modified for reduced
and OsDSM2) were simultaneously mutated for enhanced production of anti-nutrient compounds (4).
β-carotene accumulation in rice endosperm; however, no
satisfactory results were procured (101). Multiplex genome
editing was performed by targeting the OsWaxy gene at three ROLE OF GENOME-WIDE ASSOCIATION
sites for reduction of AC in rice and 14% less accumulation STUDIES IN BIOFORTIFICATION
was observed in mutants as compared to wild type (136). The
nitrogen transporter gene OsNRT1.1B, which is linked with Understanding the genetics of complex traits is fundamental
protein accumulation in rice, was edited for an increased uptake to developmental biology. Plant scientists were always

FIGURE 3 | Speedy development of biofortified crops using next generation technologies. (A) Diverse collection of germplasm, its phenotyping and genotyping for
nutrient profiling at various locations followed by genome wide association studies (GWAS) for identification of candidate genes involved in the mobility, accumulation,
(Continued)

FIGURE 3 | and partitioning of nutrients in various parts of plants. (B) Newly identified candidate genes in the previous step will be utilized in different breeding
programs using the following breeding methods such as marker assisted breeding (MAB), gene pyramiding, transgenic breeding, and CRISPR-based gene editing for
developing nutrient enriched crops. (C) Biofortified plants identified in the previous step can be continued for variety development using one of the two methods, i.e.,
conventional breeding or speed breeding. Conventional breeding will take double the number of years to achieve homozygosity in a calendar year as compared with
speed breeding. In this way speed breeding will reduce the variety development span (70).
curious to examine trait variation under different genetic micronutrients (Mn, Zn, Fe, Mg, Ca, K, N, P) and genotyped
backgrounds which have laid the foundation of different using 6,311 high-quality diversity array-derived SNPs markers.
association studies. Continued progress in genome sequencing GWAS identified 22 quantitative trait nucleotide (QTNs) for
technologies, development of high density genotyping grain nitrogen content, 5 for phosphorous content, and 1 for
arrays, and high throughput phenotyping platforms paved calcium were identified whereas no QTNs were observed for
the way for genome-wide association studies (GWAS) other micronutrients (144).
(139). GWAS starts with genotyping using different next-
generation sequencing tools, i.e., genotyping by sequencing
(GBS) or whole-genome sequencing (WGS) followed by ROLE OF SPEED BREEDING IN SPEEDY
extensive phenotyping under a given set of environments. DEVELOPMENT OF BIOFORTIFIED CROPS
The genotyping data are trimmed to obtain meaningful
SNPs which are subjected to GWAS separately for each trait The major bottleneck to the different breeding programs is
for the development of SNPs-trait association. As a result, the length of the breeding cycle of crops. After selection of
candidate genes controlling particular traits are predicted (140) parents and their intermating 4-6 generations are required to
(Figure 3). generate genetically stable homozygous lines for field evaluation,
The advent of GWAS has opened new avenues for crop which takes a further 2–3 generations and resultantly breeding
improvement by helping scientists in the identification of cycles span around 6–7 years for mono season crops and
genes controlling complex phenotypes. Different GWAS studies 3–4 years for two season crops (145). This creates demand
were conducted in cereals and pulses for the identification for novel technologies to accelerate crop growth and reduce
of genes involved in micronutrients accumulation as detailed breeding time. Breeders are already using different strategies,
below. GWAS was conducted in maize for the identification i.e., double haploid and shuttle breeding to cut the short
of genes involved in the carotenoid biosynthesis pathway. For breeding cycle of crops (146, 147). However, these methods
this purpose, 380 genotypes of maize from the CIMMYT have their limitations and speed breeding (SB) is emerging
carotenoid association mapping panel were used alongside as a novel tool for and attracting different breeders. This
476,000 SNPs markers. The key genes involved in carotenoid method was first proposed by the University of Queensland in
biosynthesis pathways in maize, i.e., DXS1, GGPS1, and 2003 as a combination of different methods to accelerate the
GGPS2 which take part in the accumulation of precursor breeding of wheat. The working principle of this method is a
isoprenoids and downstream genes HYD5, CCD1, and ZEP1 modification of the environment in such a way as to induce
which play a role in hydroxylation and carotenoid degradation, early flowering and reduce generation time (148). It applies
were identified (141). GWAS was conducted in wheat with to diverse germplasm and does not require tools for in vitro
35,648 SNPs and 123 wheat genotypes for identification of culturing and moving across the country to find a suitable
genetic regions associated with 10-grain minerals, i.e., Cd, climate for obtaining multiple generations in a year as required
Cu, Ca, Fe, Co, Li, Mg, Mn, Ni, and Zn. Ninety-two for double haploid and shuttle breeding, respectively (148)
SNPs trait association were observed among which 60 were (Figure 3).
novel and 40 were within genes. Functional annotations of It is a well-established fact that plant growth is affected
20 genes out of 40 depicted their role in grain mineral by many internal and external factors, i.e., temperature,
accumulation. The majority of SNPs were identified from D- photoperiod, light intensity, planting density, and light quality.
genome suggesting its role in controlling grain mineral diversity SB modifies these natural processes and hijacks different
in wheat (142). biological processes of plants for rapid generation enhancement.
GWAS was used to understand the genetic architecture of Knowledge of these fundamentals processes is necessary for the
seed molybdenum (Mo) and selenium (Se) in wild and cultivated development of effective SB platforms for a specific crop (148).
chickpea. For that purpose, 180 individuals including 107 wild Photoperiod plays a crucial role in the transition to reproductive
(C. reticulatum) and 73 cultivated (C. arietinum) were surveyed development by sensing any change in the external environment
using 121,840 SNPs markers and phenotyped at two locations as detected by photoreceptors. As a result, the reproductive
for 2 years. Sixteen SNPs were found associated with seed Mo success of a species is increased after synchronizing with the
and Se contents in chickpea, therefore recommending GWAS as change in external stimuli. The plants are categorized into three
a suitable technique for studying the genetics of complex traits classes, i.e., short-day plants (SDPs) which require longer than
(143). Similarly, a set of 174 accessions of Croatian common critical night length, long-day plants (LDPs) which are shorter
bean land races were phenotyped for seed contents of eight than critical night length, and day-neutral plants (DNPs) which

FIGURE 4 | Regulatory framework and risk assessment strategies for commercialization of biofortified crops developed through new breeding techniques (NBTs).
The regulatory framework consists of two methods, i.e., process-based regulation and end-product-based regulation. Whereas, risk is assessed by molecular
characterization, lab bioassays for food and for environment and NTOs. NTO, non-target organism; T-DNA, transfer DNA; GMO, genetically modified organism; SGD3,
sustainable development goal 3.

are not regulated by night length for triggering of flowering to ban genome-edited crops under GMO regulation using an
(149). Similarly, atmospheric temperature, light intensity, and unreasonable argument stating that these products are unnatural
planting density have critical roles in the modification of the and they may affect the environment (15). It has been ordered by
reproductive development of a plant. These external stimuli the European court of justice to put CRISPR edited crops under
are modified in a precise way for obtaining 4–5 generations GMO regulation which has complicated the commercialization
of a species in 1 year at the same place (145). It has and trade relation of the European and other countries’ markets.
been effectively used in many kinds of cereal and pulses for Therefore, the dependency of technology adoption and its success
shortening of breeding cycles, i.e., wheat (150), rice (151), is based on not only the evidence and scientific methods but
peas (152), chickpea (153), and many other crops. The above- also the non-government agencies, regulators, and consumers’
mentioned facts revealed that after modification of different acceptance (127, 134, 154).
genes/plant process using the abovesaid NBTs followed by rapid For transgenic crop cultivation and commercialization,
generation enhancement using speed breeding promise to deliver different regulatory processes are time-consuming and expensive
biofortified crops to consumers in shorter possible times. This with low acceptance from people toward these products.
section has also highlighted that rapid generation enhancement For example, the development of Golden Rice was a great
like speed breeding is inevitable in the days to come to ensure achievement in the field of biotechnology, but many years have
food security. passed and it is not ready for commercial cultivation because of
its unstable yield. Because of this conflict, government has not
approved its cultivation. Similarly, the cultivation of Bt brinjal
REGULATORY ASPECTS OF VARIETIES was also banned because some anti-GMO agencies, scientists,
DEVELOPED THROUGH NBTS and farmers showed concern about its cultivation. But later
on, after several tests, four more varieties were approved and
The knowledge of genomics and its implementation in plant released for commercial cultivation. Therefore, the approval of
breeding has dynamically increased the use of modern plant this technology is associated and based on public acceptance (55).
biotechnology to improve the quantity and quality of crops. New
plant breeding techniques (NBTs) are precise and accurate in
obtaining desired mutants with high target specificity. Through CONCLUSION AND FUTURE
these techniques, we can improve crops directly by deletion PERSPECTIVES
or insertion of a specific segment of a gene and ultimately
obtain desired plant traits without affecting other characters The United Nations (UN) is pushing member countries to
in a cost-effective and time-efficient manner. These NBTs can meet the 17 set SDGs by 2030. Out of these, SDG3 is about
be distinguished from other GM crops due to their stable and ensuring healthy lives and promotes well-being for all at all ages.
definite mutation. Despite its effectiveness, there is controversy Good health is tightly linked with nutrition and consumption
in many countries about its usage. Some countries like China, of nutritious food. However, hidden hunger is a major hurdle
Canada, Australia US, Brazil, and others are trying to adopt in ensuring good healthy lives by severely affecting the global
these techniques as advanced conventional breeding while other population. Most vulnerable to this issue are young children
countries like the E.U. are uncertain about their adoption and and women in developing countries. The stagnation in the
regulation (154). reduction of global micronutrient deficiency is owing to the
In the U.S. and the E.U., there are different policies for widespread use of cereals around the globe resulting in an
the production and consumption of these genetically modified imbalanced nutritional profile. Pulses along with cereals meet
crops. The main cause is people’s perception of these products. complete dietary requirements and offer a balanced diet if
Americans have a high acceptance of agriculture biotech consumed in the prescribed quantities (155). The biofortification
products while not Europeans who fear the unpredictable risk of crops apart from offering balanced nutrition to masses also
of genetically modified crops (15). The regulatory authorities, helps governments to achieve SDG3 by developing nutritionally
scientists, and policy makers are talking about the genome-edited enriched crops. Effective biofortification programs should aim
crops regulations. Their main point of discussion is whether at the development of crop varieties possessing enhanced
to put genome-edited crops under the regulatory framework of micronutrient content without compromising economic benefits
GMOs. They are recommending it on the character trait being to farmers (58). The efficiency of an effective breeding program
improved, different pathways adopted for improvement, and for biofortification is based on the availability of genetic diversity,
tools that are used, or the possible risks of crop end-products for reduction of anti-nutrients, and increased concentrations of
classification of genome-edited crops. promotor’s substances, i.e., amino acids (methionine, lysine,
In 2012 the United States Department of Agriculture and cysteine) and ascorbic acid (vitamin C) that can enhance
announced that mega-nucleases and ZNFs (Box 1) edited plants absorption of essential elements (65).
should not be placed under GMO regulation and allowed the There are certain limitations to the biofortification of crops
commercialization and cultivation of waxy corn and CRISPR through conventional and molecular means. For example, in
edited mushrooms without passing through GMO regulation conventional breeding, lack of genetic diversity in the gene pool
(Figure 4). The U.S. regulation is product-based while the E.U. is a major limitation which in some cases may be overcome
is process-based. However, some anti-GMO agencies are trying by crossing with distant relatives for the introduction of trait

of interest (TOI). However, in most cases, it is difficult to for reduced PA accumulation and maximum bioavailability of
find the desired trait in distant relatives. Hence, it is almost micronutrients (Figure 4). However, anti-nutrients compounds
impossible to improve that trait through conventional breeding, are crucial for various plant processes specifically biotic and
i.e., improving Se contents in wheat and improving oleic and abiotic stress tolerance, their knocking out will impair those
linoleic acid in the soybean (156, 157). The unavailability of TOI processes as well and there will be a need for tradeoffs (53).
in a species and its wild relatives is overcome through transgenic Another aspect of biofortification that needs consideration is
breeding. However, transgenic crops have their limitations, exploration of pathways and the role of different genes involved
i.e., low consumer acceptability and strict regulatory approval in adsorption of micronutrient from the soil, its translocation
process in various geographies. GM technology is inexpensive to the shoot, remobilization of the reserves to the reproductive
and time-consuming and is not among the good books of the part, and its bioavailability to the consumer (99). This could
current political and economic landscape. The success rate of the be achieved by using the association studies, i.e., genome wide
GM technology for cultivar released is very low irrespective of association studies which are paving the way for fast track
untiring efforts for gene identification, modification, expression identification of candidate genes involved in various metabolic
in the target organism, agronomical evaluation, and biosafety pathways (142).
assessment studies (55). As an example, we may discuss Golden As far as maize crop is concerned, three factors are of prime
Rice which was first developed and the report was published in importance concerning biofortification, i.e., quality protein
2000 and after 21 years of effort, the product is now approved maize (QPM, having high lysine and tryptophan contents),
for commercial cultivation in the Philippines (https://www.irri. pro-vitamin A, and zinc concentrations. The exploration of
org/news-and-events/news/philippines-becomes-first-country- biosynthetic pathways and genes involved thereof will help to
approve-nutrient-enriched-golden-rice) due to yield barriers unravel the genetic complexity underlying these mechanisms.
and consumer preference (118). The intervention of advanced genomics techniques, i.e., genome-
Genome editing offers a solution to most of the abovesaid wide association studies and comparative transcriptomics studies
problems. It is very precise and effective in inducing targeted for gene identification followed by modifications of key genes
modification in the gene of interest and is biologically safe as the through new breeding techniques, i.e., RNAi, overexpression, and
end product is free from transgene (133). The latest breakthrough genome editing, will open new avenues for biofortification of
in genome editing, i.e., base editing (Box 1) (the irreversible crops (44). Another important aspect concerning biofortification
conversion of a base at the target site without involving donor of crops is the utilization of crop wild relatives as source material
templates, double-stranded breaks, and dependency on NHEJ for biofortification. Different studies have highlighted that the
and HDR), prime editing (Box 1) (the introduction of indels and nutritional value of CWRs is high as compared to cultivated
all 12 base to base conversions without inducing a DNA double- crops; hence, these must be given due consideration for the
strand break using prime editing guide RNA (pegRNA) to drive transfer of desired genes using genetic engineering (Box 1)
the Cas9 endonuclease) and genome editing using rice zygote (65, 163).
(which overcomes the problem in the delivery of macromolecule Several transgenic biofortified crops have been developed
to the host cells and tissues and difficulty in transformation and are available in the research laboratories. Most of them
and regeneration) has opened new horizons for biotechnologists are not commercialized due to low consumer acceptability and
(158). As far as its regulation is considered, the U.S. and Canada regulatory issues. Further, biosafety assessment agencies and
have already declared the GE crops free from the GM regulations; regulatory bodies for GM crops demand an equal amount of
however, debate is still ongoing in the E.U. with expected positive capital as used in their development which is a big hurdle in their
outcomes (127, 159, 160). GE crops are free from transgene, commercialization. Furthermore, the consumer has concerns
hence these should be regulated like conventional breeding regarding antibiotic resistance due to bacterial markers genes
products globally and should reach a maximum consumer for present in GM crops. These issues could be tackled either through
obtaining maximum benefits of technology in a shorter possible the development of marker-free transgenic plants or the use
time (127). of genome editing tools for the development of transgene-free
The future of biofortification lies in genome editing. The plants. GE crops have to pass through a looser regulatory process
targeted area that needs to be focused on is the engineering of in some countries with the exception of the E.U. (127).
genes for increased uptake of the micronutrients from the soil, One major hurdle in the way of pulses biofortification is
maximum translocation to the seed, and increased bioavailability. that genetic variation is yet to be explored for protein and
Cereals and pulses offer a balanced diet if biofortified for Fe, Zn, starch composition and level of micro- and macronutrients.
sulfur-rich amino acids, and knocking out of anti-nutrient genes Therefore, there is a need to launch large-scale studies to
(161). The other area that needs special attention is engineering understand the genetic diversity of important macro- and
crops for reduced accumulation of anti-nutrients in cereals and micronutrients in global pulses germplasm to effectively utilize it
pulses. The highlighted example in this context is PA contents for pulses biofortification (4). The focus should be on increasing
in rice. PA is the most abundant storage form of phosphorous the bioavailability of micronutrients alongside increasing their
in plants which chelates metal ions and gets converted to concentration. For that purpose, concentration of promotor
phytate, and hence act as an anti-nutrient. Several genes involved substances (which increase absorption of minerals) should be
in the biosynthesis of PA biosynthesis (162) are identified, increased and that of anti-nutrients should be decreased. The
which needs to be suppressed through genome editing tools promotor substances are vitamin A, C, D, and E, choline, and

niacin, which increases the absorption of Se, Ca, P, Zn, Fe, FUNDING
methionine, and tryptophan (5).
Post-harvest management of crops is also in serious trouble in This study is funded by Zhejiang Province Postdoctoral Research
delivering safer foods to the consumer. Masses are unaware about Project (ZJ2020141).
dietary and nutritional significance of different parts of plants.
For example, milled grain of most cereals is consumed around
the globe. Although most of the essential elements, i.e., Se and S,
ACKNOWLEDGMENTS
are higher in germ but other essential elements, i.e., copper, zinc, We are grateful to Dr. Muhammad Zaffar Iqbal, Cheif Scientist,
and iron, are found in ample quantity in bran which is removed Ayub Agricultural Research Institute, Faisalabad and Dr.
during the milling process and is not accessible to humans (164). Sajid Ur Rahman Chief Scientist, Agricultural Biotechnology
Most micronutrients are found in the aleurone layers of the Research Institute, Faisalabad, Dr. Rana Muhammad Atif from
cereals which are removed during the milling process alongside Department of Plant Breeding and Genetics, University of
bran. As a result, these are not available for consumption. This Agriculture, Faisalabad (UAF), and Prof. Dr. Asif Ali Khan, Vice
issue needs special attention and could be tackled in two ways. Chancellor MNS University of Agriculture, Multan, Pakistan, for
Whole-grain processing is the first option that will increase the putting all their generous efforts in manuscript proofreading at
nutritional value of products obtained afterward. The second the final stages. The authors are highly thankful to colleagues at
option may be the engineering of biosynthetic pathways to alter Maize and Millets Research Institute (Yusafwala, Sahiwal) and
the deposition of micronutrients from the aleurone layer to Ayub Agricultural Research Institute (Faisalabad) for guidance
endosperm so that these may not be lost during milling and and moral support. and to Mr. Baber Ali, Mr. Ahmad Shahzad
processing (165). and Miss Sobia Jabeen Lab assistant, for technical support.
Apologies to the authors whose work has not been cited due to
AUTHOR CONTRIBUTIONS space limitations.
RS, SJ, and SA conceived the idea. RS, SJ, AN, SA, SKh, and
ZA drafted the manuscript. SA and RS prepared illustrations. SUPPLEMENTARY MATERIAL
SKa, HMUA, RAG, and WZ provided the literature and technical
assistance. RS, SJ, HMUA, SA, SKa, SKh, RAG, and WZ reviewed The Supplementary Material for this article can be found
and improved the draft. All authors contributed to the article and online at: https://www.frontiersin.org/articles/10.3389/fnut.2021.
approved the submitted version. 721728/full#supplementary-material
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published: 07 October 2021

Biofortification of Cereals and Pulses

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as well as availability of sequence genome of multiple species

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Nutrient Composition in Quantity in pulses Deficiency symptoms in humans References

Micronutrients (mg/100 g food)

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Nutrient Composition in Quantity in pulses Deficiency symptoms in humans References

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TABLE 2 | Summary of ongoing activities and achievement of HarvestPlus program.

Country Crop Nutrient Varieties developed

Pakistan Wheat Zinc Zincol-2015, Akbar-2019

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Technique Crop Micronutrient Genes involved References

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