Sundus ZAFAR, XU Jianlong

(1School of Life Sciences, Jiangsu University, Zhenjiang 212013, China; 2Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518120,China; 3The National Key Facility for Crop Gene Resources and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China)

Abstract: The nutritional quality of rice is a major concern, along with the need to enhance productivity to feed the continuously growing population. Therefore, there is a requirement to breed high-yielding rice varieties with improved nutritional quality that can help combat malnutrition, a global health issue.Undoubtedly, breeding approaches have played a significant role in increasing rice yield while enhancing its nutritional content. In addition to traditional breeding techniques, other recent approaches, such as genetic engineering, gene editing, omics methods, and agronomic practices, must also be employed to meet the nutritional needs of the current population. In this review, we offered detailed information on the development of nutritionally improved rice varieties through the enhancement of protein content, microand macronutrients, vitamins, and oil quality using genetic engineering approaches. We also identified QTLs associated with amino acids, proteins, and micronutrients in rice. Furthermore, omics approaches provide a range of tools and techniques for effectively exploring resources and understanding the molecular mechanisms involved in trait development. Omics branches, including transcriptomics,proteomics, ionomics, and metabolomics, are efficiently utilized for improving rice nutrition. Therefore, by utilizing the information obtained from these techniques and incorporating all of these recent approaches,we can effectively modify the rice genome, directly enhancing the nutritional value of rice varieties. This will help address the challenges of malnutrition in the years to come.

Key words: rice; nutritional quality; quantitative trait locus; genetic engineering; omics; gene editing

Nutrition is a fundamental prerequisite for all living beings to perform essential functions like growth,development, and production. Rice serves as a primary food source for more than three billion people worldwide, providing 20% of the world’s nutritional energy supply. In recent years, agriculture has predominantly focused on producing nutritionally improved varieties of staple crops such as rice, wheat,maize, and legumes to fulfill human nutritional requirements (Ricachenevsky et al, 2019). Developing and underdeveloped countries have increasingly emphasized the need to boost crop yield and accessibility,as these staple crops, despite being the primary source of calories, fall short in providing essential nutrients.Consequently, these countries face health issues related to nutrient deficiencies, such as anemia (iron deficiency),night blindness (vitamin A deficiency), growth retardation,and poor mental health (Oakley et al, 2004; Darnton-Hill et al, 2005). A significant portion of staple crop research explores various methods and techniques to enhance crop nutrition, encompassing both micro- and macronutrients. Rice is a noteworthy source of protein containing eight essential amino acids in balanced proportions, contributing to healthy hair, skin, heart and lung health and a well-functioning nervous system(Han et al, 2015). However, rice typically contains only

about 7% protein, which is less than what is found in pulses and wheat (Chattopadhyay et al, 2019). Despite being a major component of cereals, relatively little effort has been invested in increasing protein content in rice. Nevertheless, elevating protein levels in rice can help alleviate malnutrition in countries where rice serves as a main staple crop, meeting daily calorie requirements but lacking diverse sources of essential nutrients. Recent attention has also been given to enhancing the micronutrient content in rice grains through agronomic biofortification (Prom-u-thai and Rerkasem, 2020; Utasee et al, 2022), apart from other approaches like conventional breeding, markerassisted breeding, genetic engineering, and omics techniques aimed at improving the nutritional quality of rice. In this review, our focus is on rice quality,particularly its nutritional value and significance, and we also discuss various strategies for enhancing the nutritional quality of rice.

Rice quality characteristics and nutritional aspects

The traits and parameters used to assess rice grain quality differ across countries. However, four main quality traits are commonly used for evaluation:physical appearance, milling, cooking, and nutritional qualities (Yu et al, 2008). Achieving a meaningful increase in the economic and nutritional values of rice hinges on a comprehensive understanding of the factors governing the production of different rice varieties(Prom-u-thai and Rerkasem, 2020). Therefore, rice quality depends on numerous factors, including variety,harvesting conditions, and environmental factors such as light, temperature, humidity, and post-harvest management (Fig. 1). In this discussion, we will delve into the nutritional aspect of rice.

Table 1. Comparison of nutrient content in different staple crops.

Fig. 1. Factors affect grain quality of rice.

Rice contains a variety of vital nutrients, including calcium (Ca), iron (Fe), zinc (Zn), potassium (K),phosphorus (P), and sodium (Na), as well as vitamins and minerals crucial for human health (Birla et al,2017). However, compared with other crops such as maize, wheat, and legumes, rice tends to have lower mineral and vitamin B contents (Mahender et al,2016). A comparison of the nutrient contents of different staple crops is provided in Table 1. Brown rice is considered more nutritious than white rice because the brown layer contains protein, fatty acids,vitamins, minerals, and antioxidants (Schramm et al,2007). Rice bran, in particular, contains about 80%fatty acids. Rice oil contains unsaturated fatty acids such as oleic acid (C18:1) and α-linolenic acid(C18:3), which are essential for maintaining cell membranes and the proper function of the nervous system (Swanson et al, 2012). Despite its superior nutritional value, consumers often prefer white rice over brown rice due to its inferior texture (Mahender et al, 2016). However, the polishing process enhances the rice texture and eating and cooking quality but reduces the nutrient content of rice(Wu et al, 2013). A comparison of the nutritional value of brown and white rice is provided in Table 2.

Rice can be categorized into two types based on its color: non-pigmented rice (accounting for approximately 85% of rice) and pigmented rice(about 15%), including varieties like red, brown, black, and purple rice(Wijaya et al, 2017). Pigmented rice,such as black and purple rice,contains secondary metabolites like anthocyanin, flavonoids, and phenolics,which contribute to their status as functional foods with associated health benefits (Fitri et al, 2021;Fongfon et al, 2021). Red rice is rich in Fe and Zn,which are crucial for hemoglobin production and enzymatic processes, respectively (Boue et al, 2016).Additionally, black and red rice has been found to inhibit the proliferation of breast cancer (Ghasemzadeh et al, 2018).

Table 2. Comparison of nutritional values in brown and white rice(200 g of white rice or brown rice).

Need to improve nutritional quality of rice

Though rice is a valuable source of both micro- and macronutrients, recent climate change has reduced the nutrient content of rice and other crops (Smith and Myers, 2019). Rice grown with elevated levels of CO2has shown a decrease in vitamins, Fe, Zn, and protein contents compared with rice grown under normal CO2concentrations (Smith and Myers, 2019). Despite about half of the world’s population obtaining 25% of their calories from rice, it is estimated that increasing the protein and micronutrients could fulfill the nutritional demands of the rice-dependent population (FAO,2018). Hence, there is a pressing need to enhance micro- and macronutrients in rice and develop rice varieties with higher nutritional quality using different approaches.

Strategies for enhancing rice nutritional quality

Genetic engineering is a rapid and cost-effective method for enhancing nutrient levels in crops. Genetic engineering of rice is a potential option, as it allows for the acquisition of desirable plant traits using tissuespecific promoters, gene stacking, and the transformation and co-transformation of plasmids, enabling the transfer of multiple traits (Naqvi et al, 2009, 2010; Farré et al,2014). Additionally, genome editing has become an essential genomic tool for improving the nutritional quality of various crops. Similarly, omics approaches offer various tools and techniques to explore genetic resources and understand the genetic mechanisms involved in trait development. Therefore, incorporating omics approaches is profitable for attaining desired enhancements in rice varieties. Thus, developing superior rice varieties that can be further utilized in breeding forms the foundation of biofortification (Brar et al,2012). Fig. 2 illustrates the major strategies for enhancing the nutritional content of rice.

Genetic engineering approach for nutritious rice

Genetic engineering is considered an effective and efficient method for enhancing the nutritional quality of plants (Dias and Ortiz, 2012). Numerous efforts have been made to enhance the nutritional value of rice, as summarized in Table 3.

Proteins and amino acids

Rice protein is considered nutritionally incomplete due to its deficiency in lysine and other essential amino acids. The constitutive expression of maize lysine dihydrodipicolinate synthase (DHPS) leads to a 2.5-fold increase in free lysine in transgenic rice (Lee et al, 2001). Long et al (2013) reported an effective approach to enhance lysine concentration by expressing aspartate kinase (AK) and DHPS, while down-regulating lysine ketoglutaric acid reductase/saccharopine dehydropine dehydrogenase (LKR/SDH) expression through RNA interference (RNAi), leading to a 60-fold increase in lysine concentration in rice grains. Yang et al (2016)targeted bacterial AK and DHPS in the intragenic region of the rice genome, resulting in two pyramid transgenic lines (HFL1 and HFL2) with 25-fold higher lysine concentration in seeds compared with the wild type (WT) plants. In another approach, Liu et al (2016)over-expressed the lysine-rich protein (LRP) gene fromPsophocarpus tetragonolobusin rice using an endosperm-specific promoter, yielding transgenic rice with 30% lysine higher than control plants. Furthermore,two lysine-rich histone proteins (RLRH1 and RLRH2)were overexpressed in rice, resulting in a 35%increase in lysine concentration, along with a balanced amino acid profile as instructed by WHO dietary standards (Wong et al, 2015).

Fig. 2. Different approaches to enhance nutritional quality of rice.

Table 3. Genetic engineering approaches to incorporate nutritional genes into rice varieties.

Micronutrient manipulation

Micronutrient deficiency is a primary global health concern that has adverse effects on physical and mental development, productivity, and intellectual growth (Siddiqui et al, 2020). The insufficient intake of essential micronutrients in the human diet is often referred to as ‘hidden hunger’. This condition, along with micronutrient malnutrition, leads to over 24 000 deaths worldwide every day (Fiaz et al, 2019). The most common and severe health consequences results from deficiencies in key micronutrients, such as vitamin A, Zn, and Fe (Parashar et al, 2023).

β-Carotene

Rice cannot produce β-carotene, the precursor of vitamin A, essential for retinal function and the prevention of night blindness. Ye et al (2000)introduced golden rice (GR), also known as the prototype of GR1. They incorporated thepsy(phytoene synthase) gene from daffodils and two other genes,crtI(phytoene desaturase) andlcy(lycopene cyclase),fromErwinia uredovora. However, thepsygene from daffodils has limitations in synthesizing higher levels of carotenoids. Paine et al (2005) replaced the daffodilpsywith its homolog from maize, thereby enhancing the nutritional value of GR by increasing β-carotene content by 23-fold, with up to approximately 37 μg/g in rice endosperm. Compared with the original GR,thepsygene from maize enhances carotenoid storage,termed ‘Golden Rice 2’. Subsequently, numerous efforts have been made to develop new versions of GR (Ha et al,2010; Bai et al, 2016; Jeong et al, 2017). Tian et al(2019) expressedpsyfrom maize (ZmPsy) andCrtIfromPantoea ananatis(PaCrtI) along withtHMG1,which encodes truncated HMGR fromSaccharomyces cerevisiae. Their chemically synthesized genes (tHMG1,ZmPsy1, andPaCrtI) increase β-carotene concentration by 63-fold in rice endosperm.

Anthocyanins and folate

Anthocyanins have high antioxidant activity and are beneficial for human health. Zhu et al (2017) engineered eight anthocyanin-related genes (two maize genes and six coleus genes) with an endosperm-specific promoter,producing a novel biofortified germplasm named‘Purple Endosperm Rice’, which contains a high concentration of anthocyanins (approximately 1 mg/g).Since the human body cannot synthesize folate(vitamin B9), a folate deficiency can lead to neural tube defects, increasing the risk of cardiovascular and coronary diseases (Iyer and Tomar, 2009). Storozhenko et al (2007) enhanced the folate level in rice by overexpressing two genes fromArabidopsis thaliana,using a rice endosperm-specific promoter. The transgenic rice contained approximately 100 times more folate than the WT plants. Dong et al (2014a) evaluated the folate biosynthesis pathway injaponicarice (Kitaake)by overexpressing two genes fromA. thaliana, coding for GTP cyclohydrolase I and amino deoxychorismate synthase, resulting in a 3.2-fold increase in folate content in rice seeds.

Fe and Zn

Fe and Zn deficiencies are the most dominant micronutrient deficiencies, affecting 2 billion people globally and causing 0.8 million annual deaths(Ritchie and Roser, 2017). On average, polished rice comprises approximately 2 μg/g of Fe and 16 μg/g of Zn. Goto et al (1999) achieved a 2-fold increase in Fe content in brown rice compared with the WT by overexpressing the soybean ferritin gene (SoyferH1).Subsequently, research on Fe and Zn biofortification in rice was conducted using single-gene approaches like introducing the rice ferritin gene and soybean ferritin gene into rice (Lucca et al, 2001; Vasconcelos et al, 2003). Many approaches for Fe biofortification also lead to an increase in Zn content because most components related to Fe uptake and translocation are also linked to Zn biofortification, except for a few Fe-specific transporters like barley yellow stripe 1(HvYS1) (Banakar et al, 2017). Paul et al (2012)overexpressed rice ferritin (Osfer2), leading to a 2.09-fold increase in Fe content and a 1.37-fold increase in Zn content in transgenic rice. Furthermore,the transformation of the rice nicotianamine synthases(OsNAS3-D1) gene increases Fe content by 2.9-fold,Zn content by 2.2-fold, and Cu content by 1.7-fold compared with the controls at the seedling stage (Lee et al, 2012). To improve micronutrient content in staple crops, HarvestPlus, in collaboration with the International Rice Research Institute (IRRI), initiated a program to combat micronutrient deficiency. In 2013, they released a Zn-biofortified rice variety in Bangladesh, capable of meeting 60% of the daily Zn requirement (Goldstein, 2018). To date, the transformation of two genes (OsNAS2andSferH1) into rice produced the most promising rice variety (NASFer-274) with high Fe and Zn contents. NASFer-274 polished rice contains Fe (15 μg/g) and Zn (45.7 μg/g), exceeding the target Fe and Zn set by HarvestPlus. Additionally,no harmful effects related to heavy metal accumulation in NASFer-274 grains were observed (Trijatmiko et al,2016). Wu et al (2019) achieved Fe (12.67 μg/g) and Zn (45.60 μg/g) contents in Nipponbare, and Fe(13.65 μg/g) and Zn (48.18 μg/g) contents in IR64 using the intracellular Fe transportation gene. Both of these varieties reached the recommended Fe and Zn contents by HarvestPlus.

High nutritional oil

Rice bran oil (RBO) is extracted from rice bran and is commercially available as food-grade vegetable oil.Rice contains different concentrations of fatty acids[palmitic acid (C16:0, 18%), oleic acid (C18:1, 36%),and linoleic acid (C18:2, 37%)] that are substandard for rice storage (Tiwari et al, 2016). The shelf life of RBO is reduced due to the presence of a double bond in C18:2 (linoleic acid), resulting in a waste of 60%-70% of RBO (Chaiyasit et al, 2007). Thus, fatty acid desaturase 2 (FAD2) is suppressed by RNAi, reducing linoleic acid and enhancing oleic acid in many plants,includingArabidopsis(Lei et al, 2014) and rice(Mikami et al, 2015). Liu et al (2012a) conducted transgenic analyses of three annotated FAD genes and confirmed thatOsFAD3converted C18:2 to C18:3(α-linolenic acid, ALA). Similarly, other researchers reported a 23.8- to 27.9-fold increase in ALA content by introducing theFAD3gene into rice (Yin et al,2014). However, Cheah et al (2013) only slightly increased ALA content in transgenic rice (japonica) by overexpressing theFAD3gene. ALA has gained massive attention due to its use in producing health-promoting products and its potential to reduce the risk of cardiovascular diseases (Saravanan et al,2010; Swanson et al, 2012).

Lowering phytic acid (PA)

PA serves as the primary storage form of phosphorus(P) in cereal grains. It carries a negative charge and forms chelates with cations such as Zn, Ca, P, and Fe,forming insoluble salts referred to as phytate or phytin(Raboy, 2003). These insoluble salts inhibit the absorption of vital nutrients in the human intestine,ultimately leading to micronutrient deficiencies(Perera et al, 2018). Rice bran contains approximately 8.7% PA, prompting numerous efforts to reduce its PA levels to enhance the absorption rates of Fe and Zn(Gupta et al, 2015). In this context, research has been conducted to down-regulateOsINO1gene expression,thereby decreasing PA levels, utilizing theOlesin18(Ole18) promoters (Kuwano et al, 2009). Since the animal system lacks the phytase enzyme, it cannot digest phytate. Consequently, a low phytate rice variety has been recently developed by overexpressing theappAgene fromEscherichia coli, resulting in a 4-fold increase in inorganic P (Pi) levels, along with higher levels of Zn and Fe (Bhattacharya et al, 2019).

Undoubtedly, genetic engineering represents a potent tool for increasing the micronutrient content in rice grains. For example, successful developments include rice varieties fortified with lysine, β-carotene, anthocyanin,folate, Zn, and Fe. However, despite the scientific achievements, genetically engineered plants face a significant challenge regarding public acceptance and biosafety. Thus, various methods have been implemented to address concerns related to public safety and the potential escape of transgenes into the environment,but it increased production costs for transgenic plants(Ronald, 2014).

Gene editing for nutritious rice

Over the past decade, genetic engineering has undergone a significant transformation thanks to the CRISPR/Cas9 system, which has emerged as a groundbreaking tool for genome editing. CRISPR/Cas9 offers an effective approach that allows for the manipulation of genes at multiple genomic positions (Wang et al, 2018). Genome editing has also been employed to enhance the nutritional quality of rice, encompassing alterations in oil composition and other biofortification initiatives.

Oleic acid content

Augmenting the C18:1 content in rice at the expense of C18:2 through genetic manipulation of fatty acid profiles can elevate the nutritional value of rice, thereby enhancing its health benefits for consumers. Zaplin et al(2013) identified fourFAD2genes in rice, namelyOsFAD2-1,OsFAD2-2,OsFAD2-3, andOsFAD2-4. They elucidated the pivotal role ofOsFAD2-1in converting C18:1 to C18:2 in rice seeds and successfully generated rice varieties with elevated C18:1 content through RNAi. Tiwari et al (2016) explored the function ofOsFAD2-1in rice development via RNAi and observed its impact on several key genes in the lipid biosynthesis pathway. Abe et al (2018) employed CRISPR/Cas9-mediated targeted mutagenesis to disruptOsFAD2-1,resulting in homozygousOsFAD2-1knockout rice plants with a 2-fold increase in C18:1 content and an absence of detectable C18:2. This advancement improves the fatty acid composition of rice bran oil.

β-Carotene

Biofortification of β-caroteneis a crucial target for rice improvement. CRISPR/Cas9 was used to target theOsorgene in cauliflower, which primarily accumulates β-carotene in cauliflower. TheOsorgene, an ortholog of theOrange(Or) gene in cauliflower, was targeted in rice using CRISPR/Cas9. This genome editing resulted in enhanced β-carotene accumulation in rice(Endo et al, 2019).

Amylose content (AC)

Cereals with high AC represent a valuable source of resistant starch (Regina et al, 2006), and foods rich in resistant starch are nutritious, potentially reducing the risk of various diseases (Jiang et al, 2010). The expression levels oftwo isoforms of SBE (starch branching enzyme) genes (SBEIandSBEIIb) were simultaneously inhibited through RNAi, resulting in an increase in AC from 27.2% to 64.8%, and resistant starch from 0% to 14.6% in transgenic rice grains compared with the WT plants (Zhu et al, 2012). Later,CRISPR/Cas9 was employed for directed mutagenesis ofSBEIandSBEIIbin rice. DespitesbeI-edited plants,thesbeIIb-edited plants exhibit significantly enhanced amylose and resistant starch contents, increasing by 25.0%and 9.8%, respectively (Sun et al, 2017). Furthermore,Guo et al (2020) used CRISPR/Cas9 to induce sitespecific mutations at theSBEIIblocus injaponicarice,resulting in a 1.8-fold increase in AC and a 6%increase in resistant starch content. Additionally,nutritional quality analysis revealed significant increases in soluble sugar and lipid contents, mainly sucrose and unsaturated fatty acids, in the mutant lines. Most recently, Biswas et al (2022) adopted CRISPR/Cas9 to simultaneously target the four SBE genes in rice using the tRNA (endogenous transfer RNA) processing system to express the sgRNA (single-guided RNA) targeting the genes.SBE-edited lines exhibited a 15% increase in resistant starch content compared with the WT plants. All these findings pave the way for gene editing to enhance the nutritional quality of rice crops.Methods such as CRISPR/Cas9 circumvent regulatory mechanisms associated with genetically modified crops,addressing biosafety concerns related to transgenic crops. Currently, only a few nutrients have been targeted in rice using CRISPR/Cas9, but in the near future, the majority of nutrients should also be fortified. However, the lack of extensive knowledge about the biosynthetic pathways of essential nutrients,along with the related genes and transcription factors involved, represents some of the main challenges for further improving the nutritional quality of rice.

Omics approaches for nutritious rice

The omics approach is considered a sustainable, safe,and efficient method for crop improvement. Researchers can detect and measure various biological molecules like DNA, RNA, proteins, ions, and metabolites in living systems using omics approaches such as genetics,genomics, metabolomics, proteomics, and ionomics(Deshmukh et al, 2010; Chaudhary et al, 2015).

Transcriptomics approach

Transcriptomics studies RNA expression profiles inside the cell, addressing coding and non-coding RNA sequences. Many techniques, such as RNA sequencing and microarrays, have been developed to study gene expression in various crops under different conditions (Pandit et al, 2018). Initial efforts to study whole transcriptomes were initiated in the 1990s(Lowe et al, 2017). Using RNA-Seq, whole-genome transcription profiles ofindicaandjaponicasubspecies were obtained to understand the complexity of the rice transcriptome. The analysis of large-scale RNA-Seq data has improved rice genome annotation by discovering 1 584 novel peptides and identifying 101 new loci matched by novel peptides (Ren et al,2019). However, to date, fewer efforts have been made to explore the nutritional quality of rice using the transcriptomics approach.

Ionomics approach

Ionomics is the study of mineral nutrients and trace element compositions in diverse physiological environments and developmental phases of plants. It is an efficient omics approach that can identify genes and gene networks, regulating the ionome using techniques such as X-ray crystallography, inductively coupled plasma mass spectrometry, and neutron activation analysis(Huang and Salt, 2016). Additionally, a genome-wide association study (GWAS) was conducted on 17 mineral elements in grains from 529 rice accessions to analyze the ionome (Yang et al, 2018), providing insights into changes in mineral compositions and micronutrients across different rice accessions. Previously, a study was also performed to identify rice germplasm with increased mineral content and nutritional quality and analyze trace elements in 1 763 rice accessions(Pinson et al, 2015). Such efforts are essential for exploring genetic resources and understanding ionomic dynamics in rice tissue.

Proteomics study

Proteomics mainly explores the nutritional quality of rice by investigating the expression of bioactive compounds. Sarkar et al (2015) performed comparative proteomics to evaluate differences in the expression patterns of phenolic compounds, anthocyanins, and antioxidants in two high-yielding rice lines (KDML105 and Mali Daeng), finding that the concentrations of these compounds are higher in red rice (Mali Daeng)than in white rice (KDML105) (Maksup et al, 2018).Proteomics methods also assess changes in gene expression in transgenics due to translation activity or food nutritional quality. Ramli and Md Zin (2015)analyzed the expression levels of seed storage proteins and their association with the nutritional quality of rice varieties. They also investigated the glycomic and proteomic contents of chalky rice grains under hightemperature stress, revealing that starch degradation,rather than starch synthesis, is involved in rice chalkiness (Kaneko et al, 2016). Thus, proteome analysis,combined with crop genetics, provides insights into protein content and its genes in agronomical parts of plants under variable conditions.

Metabolomics approach

Metabolomics is a qualitative and quantitative study of small molecules in a biological system. Metabolomics analyses have revealed differences in bioactive compounds in various cooked and uncooked rice varieties. One study identified about 3 097 compounds and single nucleotide polymorphisms (SNPs) for genes regulating metabolic pathways of nutritional significance(Heuberger et al, 2010). This study investigated the effect of SNPs on rice nutritional compounds like vitamin E and phenolics, as well as changes in the metabolome of cooked rice. Likewise, metabolomics profiling of normal and giant embryo rice suggested that giant embryo rice has better quality due to its higher bioactive compounds (Zhao et al, 2019).However, the main challenge in metabolomics is extracting and interpreting vast amounts of data in a biological context.

Nutrigenomics study

Nutrigenomics is a study that investigates the effect of nutrient intake on human health, with the primary goal of enhancing food quality by improving macro- and micronutrients in cereals, fruits, and vegetables or by integrating bioactive compounds into crop plants(Tran and Kumar, 2016). Imam and Ismail (2015)investigated the nutrigenomic effects of bioactive compounds and their functional properties to identify their roles in healthy diets. However, despite being a promising avenue for personalized disease prevention,nutrigenomics faces limitations related to an individual’s dietary habits, as well as cost and timeconsuming protocols (Castle and Ries, 2009). Although the significance of nutrigenomics is widely recognized,only a few efforts have been made toward integrating it into crop improvement programs.

Omics approaches have revealed the molecular mechanisms of grain quality, contributing to the enhancement of nutritional quality. However, the genetic architecture underlying rice nutritional quality traits such as chalkiness and nutritional quality iscomplex to dissect. Currently, rational design for these traits seems complicated, and the practical implementation of these designs still needs improvement. Furthermore,this approach is expensive and requires additional support from bioinformatics platforms.

Table 4. QTLs associated with different nutritional traits in rice.

Breeding techniques for nutritious rice

Biofortification of crops through breeding involves identifying genetic resources with high nutrient content from existing germplasm. However, quality traits are polygenic and quantitatively controlled, making it challenging to improve these quality traits through conventional breeding methods. The development of genomic tools, such as molecular markers, offers practical ways to enhance the efficacy of plant breeding for transferring these quantitatively inherited traits.The application of marker-assisted selection has become possible for identifying precise genomic regions/QTLs controlling nutritional traits by adopting molecular markers (Choudhary et al, 2007).

QTLs for micronutrients

Many genetic mapping populations have been developed to dissect Fe- and Zn-related traits. Numerous new QTLs for Fe and Zn contents have been mapped using SSR (simple sequence repeat) markers in double haploid and backcross inbred line populations (Dixit et al, 2019; Islam et al, 2020; Jeong et al, 2020;Pradhan et al, 2020) as summarized in Table 4.Stangoulis et al (2007)mapped two QTLs for Zn and three QTLs for Fe on chromosomes 1, 2, 8, and 12.Garcia-Oliveira et al (2009) identified one main effect QTL for Zn, flanked by RM152 on chromosome 8.Norton et al (2010) also identified Fe (qFe-1) and other QTLs related to elements like Zn, Fe, Mn, and Ca. Dong et al (2014b) identified three QTLs for folate(qQTF-3-1,qQTF-3-2, andqQTF-3-3). Kumar et al(2014) used an F4population (579 individuals) and detected one Zn QTL and five Fe QTLs. These detected QTLs can significantly enhance the efficiency of breeding programs by improving Zn and Fe contents in rice. Swamy et al (2018) identified eight QTLs for Zn and one QTL for Fe on chromosomes 2, 3, 4, 6, 8, 11,and 12, and also detected candidate genes near Znrelated genes (OsZIPandOsNRAMP)that will help in breeding using markers to improve Zn content in rice.

QTLs for proteins and amino acids

Rice grain protein content (GPC) significantly affects the nutritional value and taste of cooked rice. About 80 QTLs for GPC have been identified and mapped on all 12 rice chromosomes. Aluko et al (2004) detected four QTLs for GPC, one of them, namedPro6, being related to theWxgene that affects rice quality (Table 4). Yang et al (2015) identified seven GPC QTLs in three locations using chromosome segment substitution lines, and onlyqPC-1was consistently found in all three locations. Chattopadhyay et al (2019) used a 40 K Affymetrix custom SNP array and detected three QTLs for GPC (qGPC1.1,qSGPC2.1, andqSGPC7.1).Several QTLs for protein content have been identified in rice, includingqPro-1,qPC1.2, andqPC1on chromosome 1, andqAAC7.1,qPC6.2, andqPro-2on chromosomes 7, 6, and 2, respectively (Kinoshita et al,2017; Islam et al, 2020; Jang et al, 2020). Numerous studies have used linkage methods to improve rice amino acid compositions by providing genetic information from different mapping populations. Yoo(2017) mapped six main effect QTLs located on chromosome 3, contributing to 10.2%-12.4% of phenotypic variations for amino acids. The QTL cluster (qAla3,qVal3,qPhe3,qIle3, andqLeu3) was associated with five amino acids. These results are valuable for marker-assisted breeding programs and the identification of candidate genes that enhance amino acid content in rice. It is also possible to phenotype the biochemical attributes of rice using hyperspectral techniques and this data for GWAS. The normalized difference spectral index (NDSI) highly correlates with protein content. Based on GWAS analysis, NDSI identified all 43 genes and annotated 3 pathways that are precisely the same as protein content,which is essential for the biosynthesis of some amino acids/proteins in rice grains (Sun et al, 2019). This study offers a new approach to phenotype important biochemical traits for determining rice quality that could be used in genetic studies.

QTLs for PA

Significant reductions in nutrient element bioavailability have been observed due to PA in the form of phytate(Gemede, 2014). Liu et al (2005) identified a broad range of PA contents, ranging from 0.685% to 1.030%,by assaying 72 rice varieties. They also observed the major effects of varieties, environments, and their interactions on PA. While Liu et al (2007) reported genetic variation for PA, Stangoulis et al (2007) detected two QTLs for grain phytate from an IR64 × Azucena double haploid population. Mn, Fe, and Zn contents showed different genetic regulations due to their locations on other chromosomes compared with the phytate position. Thus, there is an opportunity to discover segregants containing high Fe, Zn, and Mn and low PA contents. The QTLs detected in these studies can be valuable in improving rice nutritional quality.

In recent years, significant advancements have been made in genetic studies on the levels of Zn, Fe,vitamins, minerals, proteins, and amino acids, and QTLs linked to these traits. However, further research is needed on processing and curative properties. The availability of gene-based markers and other advanced tools will assist breeders in selecting specific gene alleles that play a critical role in the nutritional traits of rice. Although breeding methods can be resourceintensive, their benefits have proven to make them cost-effective. Thus, effective steps such as increased financial assistance, training of plant breeders and technicians, improved infrastructure, and the availability of raw materials should be taken to facilitate the adoption of breeding methods worldwide.

Agronomic biofortification

Apart from breeding approaches and transgenic methods,agronomic biofortification is a technique based on applying fertilizers to enhance the nutritional quality of rice. Generally, plants absorb micronutrients from the soil, and applying fertilizers in the soil and foliar methods can address the deficiency of micronutrients in plants (Garg et al, 2018). An agronomic practice was conducted in five countries to enhance the contents of Zn, iodine (I), Fe, and selenium (Se) in rice grains through foliar application. On average, brown rice increases Zn (21.4-28.1 mg/kg) and I (11.0-204.0 mg/kg) by foliar application of Zn and I. In contrast, a 26.8 mg/kg increase in Zn content and a 181.0 mg/kg increase in I content were reported with the cocktail application (Zn + I + Fe + Se) (Prom-u-thai et al,2020). Furthermore, high Zn accumulation also increases Fe accumulation and reduces PA content(Saha et al, 2017). Different locations can also alter the levels of micro- and macronutrients in rice by application of fertilizers (Chandel et al, 2010).

Agronomic biofortification of crops with Fe, Se,and Zn using organic fertilizers holds remarkable potential in addressing nutrient deficiencies. However,the success of agronomic biofortification depends on various factors, such as field experiments, the influence of soil properties on micronutrients, and the responses of crops or different varieties of the same crop on Zn,Se, and Fe fertilization. These factors still need to be explored in detail to achieve the desired results.

Conclusions and future prospects

Enhancing the nutritional quality of crops is a promising and cost-effective agricultural strategy for improving the nutritional status of undernourished populations worldwide. However, crop biofortification presents significant challenges. Therefore, there is a need to combine recent technologies such as plant breeding, genetic engineering, omics techniques, and agronomic approaches. Breeding approaches have been widely used due to their high success rate and acceptance. While transgenic plants have also shown promise, they face challenges such as cost, public acceptance, and lengthy approval processes in various countries. Despite these challenges, biofortified crops offer great potential to combat micronutrient malnutrition,especially in developing countries. In recent years,genome editing research has made significant progress in enriching food crops with higher nutritional value,offering a bright future in addressing malnutrition challenges. Additionally, the integration of omics tools,including transcriptomics, metabolomics, proteomics,and ionomics, is pivotal in developing highly nutritious rice varieties. However, there is a widening gap between the development of omics resources and their effective utilization, requiring more efforts to bridge this gap. Recent advancements in genetic engineering,agronomic biofortification, and omics approaches can complement breeding programs and improve the nutritional quality of rice.

ACKNOWLEDGEMENTS

This study was funded by the Hainan Yazhou Bay Seed Lab Project, China (Grant No. B21HJ0216), the Key Research and Development Project of Hainan Province, China (Grant No.ZDYF2021XDNY128), and the Agricultural Science and Technology Innovation Program and the Cooperation and Innovation Mission, China (Grant No. CAAS-ZDXT202001).