The rice grain can be divided into two major parts: glume (rice husk) and caryopsis (brown rice). Rice glume, including the inner glume, outer glume, lodicule, and tip of the glume (elongated to form an awn), consists of four parts. The fruit obtained after removing the inner and outer rice glumes is the caryopsis (i.e., brown rice). The caryopsis is composed of three parts: cortex, endosperm, and embryo (Dou et al., 2018). The weight proportion of each rice component and brown rice grains varies considerably due to the different types and varieties of rice, soil, climate, and cultivation techniques. As the protective tissue of the endosperm, the rice husk accounts for 18–20% of the rice, has high contents of crude fiber and combined minerals (silicon), and a hard texture. The pericarp and testa account for 1.2–1.5% of the rice grain, the aleurone layer accounts for 4–6% of the rice grain, the endosperm accounts for 66–70% of the rice grain, and the embryo accounts for 2–3.5% of the rice grain (Wu et al., 2016).

Rice mainly contains carbohydrates, moisture, proteins, lipids, minerals, and vitamins (Safdar et al., 2023). The nutritional components of polished rice are far less than those of brown rice (brown rice is obtained after removing the rice husk). Functional components of rice are divided into nine major categories, including functional proteins (low-content glutelin, 26-kD anti-allergic protein), active polysaccharides (resistant starch, dietary fiber, and its analogs), functional oils (rich in unsaturated fatty acids), functional vitamins (β-carotene), essential trace elements (iron, zinc, and selenium), functional flavonoids, free radical scavengers, functional peptides, and essential amino acids for the human body (Su et al., 2007). The exploration and utilization potential of functional rice components is huge. For example, functional rice components can enhance human body functions and metabolic balance, thereby increasing the added value of rice and expanding the utilization range of rice. This makes rice not only food for human beings, but also an important raw material for new functional health products and the food industry.

The classification of functional nutritional rice is diverse, and each classification method provides a mechanism of understanding, creating, and applying functional nutritional rice more extensively (Jin and Nie, 2023). With the constant advancement of scientific research technology and people’s higher pursuit of a healthy diet, the classification and application of functional nutritional rice is anticipated be more in-depth and extensive. This article summarizes the existing common types of functional nutritional rice, and the corresponding varieties and germplasm information list ( Table 1 ) (Hu and Hu, 2021; Liu et al., 2024).

Starch synthesis in the endosperm begins with the photosynthetic production of glucose, which is then transported into plastids by transporter proteins and converted into ADP-glucose by the enzyme ADP-glucose pyrophosphorylase (Kammerer et al., 1998; Bouis et al., 2011). ADP-glucose is used to synthesize amylose by granule-bound starch synthase and amylopectin synthesized by the coordinated action of soluble starch synthase, starch branching enzyme (SBE/BE), and starch debranching enzyme (Smith, 2008). Regular rice has a substantially low resistant starch content of <1%. Increasing resistant starch content in rice can effectively reduce starch digestion rate, increase satiety, and prevent various diseases, such as diabetes and obesity (Ballicora et al., 2003). This is a crucial direction for rice quality improvement (Sharma et al., 2008).

The formation of resistant starch in rice is regulated by the soluble starch synthase gene SSIIIa and granule-bound starch synthase I (Waxy). Knocking out SSIIIa promotes accumulation of resistant starch in rice (Yadav et al., 2010). Knocking out SSIIIa (SS3a) and SSIIIb (SS3b) genes in Nipponbare increased resistant starch content in SS3a mutants from 0.58% in the wild-type rice to approximately 5%; however, no significant differences were observed in the resistant starch content of SS3b mutants. However, resistant starch content in the double mutants (ss3a ss3b) further increased to 9.5% (Zhou et al., 2016). Editing SS3a and SS3b genes in Zhonghua 11 has also yielded similar results (Huang et al., 2024). The absence of SBEI and SBEIIb genes in rice results in increased resistant starch accumulation (Tsuiki et al., 2016; Guo et al., 2020; Wang et al., 2023). Inhibiting the expression of SBE encoding genes SBEI and SBEIIb increases resistant starch content in rice to 14.9% (Wei et al., 2010; Chen et al., 2022). Miura et al. crossed natural mutants of SBEI and SBEIIb to develop a high-resistant double mutant rice (seb1 seb2b), which had a resistant starch content of 35.1% in the endosperm (Zhu et al., 2012; Miura et al., 2021).

The accumulation of anthocyanins/proanthocyanidins in the seed coat confers brown rice its distinctive color and certain nutritional benefits (Xu et al., 2015). Red rice contains only proanthocyanidins, while black and purple rice contain both anthocyanins and proanthocyanidins (Finocchiaro et al., 2010). The red seed coat of rice is controlled by the complementary interaction of two major genes, Rc and Rd. The rice seed coat appears red when both Rc and Rd are present; the seed coat appears brown when only Rc is present; and the seed coat appears white when Rc is mutated to rc (Furukawa et al., 2007). The purple seed coat of rice is regulated by two dominant complementary genes, Pb and Pp. The rice seed coat is white when Pb is absent; the seed coat is brown when only Pb is present; and the seed coat is purple when both Pb and Pp are present (Rahman et al., 2013). Nutritionally enhanced rice germplasm has been developed using endosperm-specific promoters to introduce metabolic pathways of secondary metabolites, such as astaxanthin, anthocyanins, and β-carotene into rice. These genetic introductions result in the display of various colors, such as red, purple, and yellow by rice seeds. Astaxanthin is biosynthesized from β-carotene by β-carotene ketolase (BKT) and β-carotene hydroxylase (BHY) (Higuera-Ciapara et al., 2006). The initial precursors of carotenoids (including β-carotene) are synthesized via the 2-C-methyl-D-erythritol-4-phosphate pathway (Zhu et al., 2009). Biofortified rice germplasm with high anthocyanin content in the endosperm has been developed by simultaneously transforming rice with genes, such as MYB, bHLH, HY5, and WD40. Rice with endosperm rich in astaxanthin has been successfully developed (Zhu et al., 2009) by introducing genes, such as sZmPSY1, sPaCrtI, sCrBKT, and sHpBHY. The first generation of Golden Rice with β-carotene-rich endosperm was developed (Zhu et al., 2017) by introducing the phytoene synthase (PSY) gene from daffodil, lycopene β-cyclase (LCY) gene, and carotene desaturase (CRTI) gene from bacteria into rice (Beyer, 2010). Subsequent modifications by introducing the PSY gene from maize and CRTI gene from bacteria into rice further increased β-carotene content by nearly 23-fold (Ye et al., 2000).

The embryo of giant embryo rice is two to three times larger than that of regular rice and several genes regulating rice embryo size have been identified. The giant embryo (GE) gene was the first to be identified in rice (Zhu et al., 2018). GE in rice encodes a P450 protein that is a member of the CYP78 family. The indole-3-acetic acid content in the rice grain of ge mutants reduced considerably, thereby affecting the expression of genes involved in the cell cycle. Loss of GE function results in an enlarged embryo and a reduced endosperm (Yang et al., 2013). Other than GE cloning, mutations in the Goliath (GO), PLASTOCHRON3 (PLA3), and LARGE EMBRYO (LE) genes have resulted in enlarged embryos, with PLA3 and GO being the same gene. PLA3/GO encodes a glutamate carboxypeptidase II, which is a member of the M28 family of metalloproteases (Nagasawa et al., 2013). LE is the first C3HC4-type RING finger protein reported to be involved in embryo morphogenesis. Inhibition of LE gene expression by RNAi technology results in an enlarged rice embryo, although the specific molecular regulatory mechanism remains unclear (Taramino et al., 2003).

Protein is the second largest storage substance in rice endosperm, predominantly consisting of four components: glutelin, prolamin, globulin, and albumin. Glutelin has the highest content, accounting for approximately 60–80% of the storage proteins (Lee et al., 2019). The eating quality of rice varies with similar amylose content and a low protein content enhances the eating quality of rice. Therefore, reducing protein content is an effective strategy for improving the eating quality of rice (Saito et al., 2012; Yang et al., 2019). Studies have revealed that mutations in the LGC1 gene on chromosome 2 of rice can reduce glutelin content in grains to less than 4% (Miyahara, 1999). Amino acid transporter encoding genes OsAAP6 and OsAAP10 have been edited using CRISPR/Cas9 technology, and mutations in OsAAP6 and OsAAP10 substantially reduce rice protein content and improve taste scores (Miyahara, 1999; Longchamp et al., 2015). Rice glutelin synthesis genes are divided into four subfamilies: GluA, GluB, GluC, and GluD, which are directly related to rice protein content and taste quality (Takahashi et al., 2019; Wang et al., 2020). Simultaneous knocking out of eight highly expressed glutelin family genes (OsGluA1, OsGluA2, OsGluA3, OsGluB2, OsGluB6, OsGluB7, OsGluC, and OsGluD) using CRISPR/Cas9 technology resulted in seven different mutant combinations. Consequently, the protein content of rice decreased to varying degrees, and the hardness, appearance, and taste value of rice improved considerably (Takahashi et al., 2019). New rice germplasm with reduced glutelin content has been developed using CRISPR/Cas9 technology by knocking out multiple glutelin encoding genes (OsGluA1, OsGluA2, OsGluA3, OsGluB1a, OsGluB1b, OsGluB2, OsGluB4, and OsGluB5) (Yang et al., 2022).

Over a billion people worldwide have iron and zinc deficiencies. Enriching rice grains with iron and zinc is considered the most effective method of addressing these nutritional deficiencies (Wessells and Brown, 2012; Chen et al., 2022). The pathways of iron and zinc absorption, transport, and storage in rice have been extensively studied. Biofortification can be achieved through various methods, such as increasing the binding of iron/zinc with phytosiderophores and plant iron carriers at the root zone, in turn, enhancing iron/zinc absorption, promoting long-distance transport of iron/zinc within the plant, increasing endosperm-specific storage of iron and zinc, and altering the expression of transcription factors ( Table 2 ).

The iron-regulated metal transporter 1 (IRT1) gene encodes iron (II) and zinc (II) transporters in plant roots. Overexpression of OsIRT1 or AtIRT1 in rice substantially increases the accumulation of iron and zinc in rice grains (Lee and An, 2009; Miller, 2013). The yellow stripe 1-like gene 15 (OsYSL15) is associated with the uptake of Fe(III)-DMA. Similarly, overexpression of OsYSL15 increases the iron content in mature rice seeds by approximately 1.2-fold (Lee et al., 2009a). Phytosiderophores and nicotianamine are chelators involved in the internal transport of metals in plants. These chelators are crucial for iron absorption in rice and possibly facilitate zinc absorption. Studies have shown that overexpression of nicotianamine synthase genes OsNAS1, OsNAS2, and OsNAS3 in rice considerably increases iron and zinc contents in rice grains (Lee et al., 2009b; Lee et al., 2011; Lee et al., 2012; Boonyaves et al., 2016). Similarly, introducing the barley gene HvNAS1 into rice or simultaneously transferring several different NAS genes from rice and barley into rice increases iron and zinc contents in rice (Masuda et al., 2009; Johnson et al., 2011). Specific expression of the ferritin-encoding gene in the rice endosperm is an effective strategy for increasing iron content in rice grains (Lucca et al., 2001b; Banakar et al., 2017). Regulating the expression of soybean FER (GmFER) or common bean FER (PvFER) genes through endosperm-specific promoters substantially increases endosperm iron content (Goto et al., 1999; Banakar et al., 2017). Arabidopsis FERRIC REDUCTASE DETECTIVE 3 (AtFRD3) is a transporter that loads citrate into the xylem of root columns, thereby increasing iron mobility within plants (Durrett et al., 2007). Iron and zinc contents increased by 5.4- and 2.4-fold, respectively in the polished grains of transgenic rice lines with constitutive expression of AtFRD3 when compared to those of control plants (Wu et al., 2018a). Additionally, pOsSUT1 plants with polished grain iron content 4.4-fold higher than those of wild-type plants have been developed through the expression of the iron(II)-NA transporter encoding gene OsYSL2 under the control of the rice SUCROSE TRANSPORTER1 promoter (pOsSUT1) (Ishimaru et al., 2010). Vacuolar membrane transporter 2 (OsVIT2) encodes an iron and zinc transporter on the vacuolar membrane. Knocking out OsVIT2 reduces the accumulation of iron and zinc in the flag leaves and increases their transport to the sink tissues, which directly leads to high iron and zinc contents in rice seeds (Bashir et al., 2013). Studies have shown that OsYSL9 can transport Fe-NA/DMA complexes from the endosperm to the embryo during seed development. Knocking down OsYSL9 increases the endosperm iron content by more than twofold (Senoura et al., 2017). The iron content of brown rice increased by more than twofold through overexpression of the transcription factor IRON‐RELATED BHLH TRANSCRIPTION FACTOR 2 (OsIRO2), which is involved in iron deficiency responses in rice (Ogo et al., 2011). Introducing multiple genes simultaneously can produce stronger biofortification effects than single gene transformations that produce weak biofortification effects. For example, introducing GmFER, HvNAS1, and OsYSL2 simultaneously increases iron and zinc contents in polished rice by 6- and 1.6-fold, respectively (Ogo et al., 2011). Introducing OsNAS2 and GmFER into rice increases iron and zinc contents in polished rice by 7.5- and 3.5-fold, respectively (Masuda et al., 2012). Simultaneous introduction of AtFRD3, AtNAS1, and PvFER increases iron and zinc contents in polished rice by 5.4- and 2.4-fold, respectively (Durrett et al., 2007).

Selenium is an essential microelement for humans and its deficiency can lead to potential health risks. Studies have shown that different genotypes of the same plant species exhibit marked differences in selenium absorption. Selenium and sulfur belong to the same group, and have similar chemical structures and properties, suggesting that both elements can enter plants through the same metabolic pathway (Trijatmiko et al., 2016). Numerous proteins involved in selenium absorption and transport in rice have been reported. For example, SULTR1;1 and SULTR1;2, which are potential sulfate transporters, thereby facilitating sulfur absorption (Sun et al., 2021). Furthermore, overexpression and knockout of OsPT2 in plants considerably increases and decreases selenite uptake, respectively, indicating that OsPT2 is involved in selenite uptake (Zhang et al., 2014). Overexpression of OsPT8 substantially increases selenium content in tobacco plants, indicating that OsPT8 is also involved in selenite absorption and transport (Song et al., 2017). Yeast expressing OsNIP2;1 enhances selenite uptake at pH 3.5 and 5.5, but not at pH 7.5 (Zhao et al., 2010). Overexpression of NRT1.1b considerably increases selenium content in both rice stems and grains (Zhang et al., 2019). A mutation of the 566^th base of the OsASTOL1 gene from G to A in rice resulted in the change of the 189^th amino acid from serine to aspartate, in turn, producing the OsASTOL1^S189N gain-of-function mutant. This mutant not only reduces arsenic accumulation in rice grains but also increases selenium content by more than onefold (Zhang et al., 2014).

In conclusion, with the advancement of biotechnology and in-depth study of functional genes, numerous gene functions have been discovered and reported. The application of technologies, such as CRISPR/Cas9, RNAi, and gene overexpression to modify rice metabolic pathways and enrich nutrients in the rice endosperm is an effective approach for biofortification. However, the practical application of these technologies is associated with challenges, such as the safety assessment of gene-editing technologies, stability of nutrients in rice, and how to improve consumer acceptance.

Conventional breeding falls under the breeding 2.0 era, which is a breeding era characterized by experimental design, field statistics, and analysis (Wallace et al., 2018) Chinese rice breeder Huang Yaoxiang, who was internationally known as the “father of semi-dwarf rice”, developed the Guangchangai rice variety through hybrid breeding in 1959. Subsequently, other breeders bred dwarf varieties, such as Aijiaonante and Dee-geo-woo-gen. The dwarf rice variety is resistant to lodging and fertilizer, and has a high yield (Tu and Wang, 2019). Chinese breeders pioneered the first revolution in rice breeding and made substantial contributions to the first Green Revolution (Zheng et al., 2024). The IR8 developed by the IRRI in 1966 and was bred using Dee-geo-woo-gen, has become an internationally recognized landmark achievement in the “Green Revolution” and the first breakthrough in the history of rice breeding (Zheng et al., 2024). From 1962 to 1988, IRRI developed a total of 34 rice varieties, with their selection being achieved through conventional breeding. Most of the varieties have good appearance quality, high yield, high rice quality, and strong stress resistance, and they have been introduced to several countries globally for planting and promotion. The conventional breeding of functional nutritional rice, which is often achieved through hybridization and backcrossing based on the appearance phenotype, relies on the germplasm resources of functional rice. For example, colored rice such as black, purple, red, and green rice are easy to distinguish in appearance and can be directly selected for breeding with relatively high efficiency. In addition, even though several biofortified rice varieties for protein and zinc have been developed in Asia (Hu and Hu, 2021; Wu et al., 2022). the selection of fragrant rice germplasm is determined by sensory evaluation; that is, smelling and chewing. However, the disadvantage is that sensory evaluation is limited to rice varieties that are easily recognizable in appearance.

In 1953, the discovery of the DNA double helix model marked the arrival of the era in which molecular biology (Watson and Crick, 2003) and genetics research was advanced to the molecular level and crop breeding was ushered into the era of molecular breeding. Molecular breeding, also known as breeding 3.0, is a combination of modern biotechnology and conventional breeding methods guided by theories, such as genetics and molecular biology, to achieve an organic combination of phenotype and genotype selection, and cultivate excellent new varieties (Wallace et al., 2018). In conventional plant breeding, genetic variation is primarily identified through observation and measurement. The development of molecular biology has enabled researchers to detect variations in DNA sequences that can affect phenotypic traits and DNA sequence variations can be detected using various techniques. Molecular marker technology, which confers the advantages of abundant quantity, site specificity, dominant or codominant, high resolution, and good stability (Jander et al., 2002), has gradually developed. Some biologically active substances or special components in rice cannot be identified through phenotypic observations. Based on the identification of key genes involved in the synthesis or metabolism of biologically active substances, molecular markers that are closely linked to such genes can be used to aggregate multiple traits into one variety. A combination of molecular markers and conventional breeding has the advantages of clear objectives, high accuracy, no environmental interference, and an accelerated breeding process (Jin and Nie, 2023). Although most genes regulating crucial agronomic traits have been identified in rice, few genes with nutritional value and unique biologically active ingredients have been identified ( Table 2 ). Moreover, fewer genes can be used for molecular marker-assisted selection, which considerably limits the widespread application of functional rice breeding. At the same time, the play of some key regulatory genes requires a specific genomic background. Therefore, in addition to the selection of key genes, genome selection must be carried out.

Mutation breeding refers to the use of chemical, physical, and other factors to induce mutations in organisms under human conditions. Grain crops have regularly been the focus of mutagenesis breeding and according to statistics, the number of varieties bred through mutagenesis accounts for approximately 10% of the total number of varieties bred during the same period. For example, an early Chinese Indica rice variety called Yuanfengzao was developed using IR8 and 60Co radiation treatment, followed by mutagenesis selection (Zhang and Tai, 2001).

Functional nutritional rice can be obtained by mutagenesis screening and identification of specific functional nutritional components. For example, the world’s first giant embryo rice “Haiminori” ( Table 1 ) was bred by crossing the giant embryo mutant EM40 that was selected by chemical mutagenesis of Kinmaze with the high-yielding variety Akenohoshi ( Table 1 ). The embryo volume of the giant embryo rice is three- to fourfold that of ordinary rice (Maeda et al., 2001). Low glutelin content-1 (LGC-1) rice cultivar was developed through chemically-induced mutation of Nihonmasari, a high-quality rice cultivar from Japan to obtain a mutant NM67 with substantially reduced endosperm glutelin content. The mutant was then backcrossed with the recurrent parent Nihonmasari for the first time to obtain LGC-1 (Lida et al., 1993). Compared to conventional rice, LGC-1 rice has low glutelin content (<4%), increased prolamin content, and considerably reduced total absorbable protein, which can be consumed by patients with diabetes (Kusaba et al., 2003). LGC1 is a key germplasm resource for breeding functional rice and is widely used by breeders. Low glutelin protein cultivars, such as LGC-Katsu and LGC-Jum, have been developed successively using LGC-1 as a parent (Nishimura et al., 2005).

The major challenge associated with mutagenesis breeding is the low frequency of beneficial mutations, as well as difficulties in orientation and nature of mutations. Therefore, it is essential to improve mutagenesis efficiency, rapidly identify and screen mutants, and explore targeted mutagenesis pathways. Mutation breeding should be combined with other breeding technologies to improve breeding efficiency. Wan Jianmin from Nanjing Agricultural University in China introduced the LGC-1 rice cultivar and its LGC1 gene into the dominant rice varieties. Through hybridization, backcrossing, and multiple crossing, combined with phenotype identification, a broad range of low glutelin protein content rice varieties, such as W3660, W204, W1721, W0868, and W088 have been bred successively using molecular marker-assisted selection. The low glutelin protein content of rice has decreased considerably and globulin is deficient, thereby resulting in a further decrease in its absorbable protein content and postprandial blood glucose levels. The applicable population has further expanded, and commercial planting and promotion of this rice variety has begun (Wan et al., 2004; Chen et al., 2023).

Genetic engineering technology was marked by the establishment of DNA recombination technology in the 1970s (Cohen et al., 1973) and was used in the 1980s for transgenic plants, also known as genetically modified (GM) plants. Genetic engineering technology refers to the use of genetic manipulation methods to develop plants with new superior and artificially-designed traits. GM breeding has been the fastest-developing field of agricultural biotechnology application since the 1980s. The cumulative planting area of GM crops worldwide between 1996 and 2019 was 2.7 × 10⁹ hectares (hm²) (Li et al., 2023). A GM tomato with delayed ripening (Flavr Savr) was approved for consumption on May 18, 1994. The Flavr Savr tomato was the first GM tomato approved for commercial cultivation by the Food and Drug Administration of the United States, and its acceptance among consumers was positive (Kramer and Redenbaugh, 1994). The accumulation of specific biologically active substances in the endosperm to enhance the nutritional quality of rice can be achieved by expressing and modifying key genes involved in the synthesis of biologically active substances in rice or restructuring the synthesis pathways of certain biologically active substances that cannot be directly synthesized in the endosperm. The endosperm of rice seeds is an ideal bioreactor for metabolic engineering and food bioenhancement (Zhu et al., 2017), such as the first transgenic of rice developed for high iron content by overexpression of OsNAS1 (Lee et al., 2009b). Currently, various nutrient-enriched rice varieties have been developed based on these strategies. The common target micronutrients obtained through bioenhancement breeding are iron, zinc, vitamins, flavonoids, and carotenoids. Most of the genes related to carotenoid synthesis in the rice endosperm are either unexpressed or exhibit low expression. Anthocyanins and carotenoids have become popular research directions when compared to other bioactive compounds due to their distinct synthetic pathways.

Golden Rice is a new type of GM rice with three newly inserted coding genes, which express PSY from daffodil, LCY, and CRTI from the soil bacterium Erwinia uredovora that regulate β-carotene synthesis in the rice endosperm. Golden Rice is rich in carotene, which can be converted into preformed vitamin A in the human body (Ye et al., 2000; Beyer, 2010). Astaxanthin is a red-colored xanthophyll carotenoid that is essential for maintaining organism balance and reducing the accumulation of senescent cells. However, astaxanthin is only produced by some algae and yeast, and cannot be produced by most higher plants. Therefore, ways of obtaining astaxanthin, which has strong antioxidant properties, from the daily diet of humans are few. A research team led by Liu Yaoguang from South China Agricultural University in Guangzhou, Guangdong, China has developed a synthetic metabolic engineering strategy and a multigene stacking system TGSII using rice endosperm-specific promoters. The researchers combined astaxanthin with the common grain crop rice to develop “Purple Endosperm Rice” that has a high anthocyanin content (Zhu et al., 2017). Four key genes involved in carotenoid biosynthesis, namely phytoene synthase (sZmPSY1), phytoene desaturase (sPACrTI), β-carotene ketolase (sCrBKT), and β-carotene hydroxylase (sHspBHY) genes have been expressed in rice using the TGSII system (Zhu et al., 2017). Different combinations of carotenoid/ketocarotenoid/astaxanthin biosynthetic pathways were reconstructed in the rice endosperm, resulting in purple embryo rice with astaxanthin exhibiting high antioxidant activity and removal of the transgenic screening marker. The GM rice developed using this technology are Canthaxanthin and Astaxanthin rice. The study used exogenous genes to compensate for the endogenous astaxanthin synthesis pathway in rice, in turn, leading to astaxanthin synthesis in the rice endosperm for the first time (Zhu et al., 2018). Enhancing the expression of related transporter genes can improve the absorption and utilization efficiency of iron and zinc in plants, which is an effective transgenic strategy for increasing crop iron and zinc contents (Blancquaert et al., 2015). Moreover, the coexpression of lactoferrin (iron chelating glycoprotein) and ferritin genes increases iron content in crops (Goto et al., 1999; Drakakaki et al., 2000). Simultaneous expression of genes encoding ferritin and nicotianamine synthase in crops increases zinc and iron contents (Wirth et al., 2009). Numerous biofortified crops with high iron and zinc contents, such as rice, corn, and wheat (Kumar et al., 2019), have been developed using genetic engineering technology. Crops biofortified with nutrients have a great potential to address global mineral deficiencies (Jin and Nie, 2023; Liu et al., 2024).

Genome editing technology is a breakthrough technology in the field of biological genetic manipulation after transgenic technology. The CRISPR/Cas system is a novel genome editing technology that emerged after the zinc finger nuclease and transcription activator-like effect nuclease technologies. This technology involves site editing of specific genome sequences using a guide RNA and the cooperating nuclease Cas9, and has the advantages of simplicity and efficiency (Mahfouz et al., 2011; Jinek et al., 2012). The CRISPR/Cas9 system has enabled genome editing in various crops, such as rice, wheat, corn, soybean, rapeseed, tomato, watermelon, strawberry, and it is at the forefront of research in rice and wheat, thereby providing important technical support for crop variety improvement and new variety cultivation.

A japonica rice variety cultivated using LGC-1 as the donor parent was used as the transgenic recipient. The starch branching enzyme IIb (SBEIIb) gene was knocked out using CRISPR/Cas9 technology to obtain a high-resistance starch and low glutelin protein mutant line without transgenic components. The apparent starch, resistant starch, and glutelin contents of the mutant line increased by approximately 1.8-fold, 6%, and 2% (Tsuiki et al., 2016; Guo et al., 2020), respectively. Chen et al. (2022) used CRISPR/Cas9 technology to simultaneously edit nine glutelin genes in LGC germplasm and obtained nine homozygous edited strains without T-DNA (Chen et al., 2022). Due to different regulatory policies on gene editing in various countries, the cultivation of functional nutritional rice bred using CRISPR/Cas9 is still at the laboratory and publication stage. Solving specific regulatory challenges or consumer safety concerns is a complex problem. Due to length limitation and different focus, this review cannot discuss all the concerns you have raised. However, in the revised manuscript, we raised these questions to draw attention from all aspects.

From the perspective of cultivated varieties, the breeding of functional nutritional rice predominantly involves the aforementioned five aspects. However, currently, several countries in the world, including China, have regulations on the management of GM crops. Genetic engineering and gene editing products cannot be directly planted or consumed at this stage. In the actual breeding process of functional nutritional rice, multiple methods are often used in combination to accelerate the research and development process of functional rice. Although some progress has been made in molecular breeding, mutagenesis, genetic engineering, and gene editing technologies, the maturity of these technologies, especially in identifying and utilizing functional rice germplasm resources, still needs to be improved. Because the entire experimental process is still time-consuming and costly, it limits its widespread commercial application. The implementation of these technologies highly relies on advanced scientific instruments and relatively complex technical means, which limits the promotion of the technology. Due to the limited investment in research and development, the gap between developing countries and developed countries may last for 20-30 years. Moreover, research on the genes and molecular mechanisms related to functional nutritional rice is still very limited due to the pleiotropy of genes. Therefore, many unknown factors are still unpredictable when using these technologies for genetic improvement of functional nutritional rice. Especially in gene editing breeding, due to patent protection of core technologies, the discovery and identification of excellent genes in germplasm resources are lagging behind, which affects breeding efficiency and accuracy. In addition, due to the conservative safety evaluation and approval processes for genetically engineered rice in different countries, the commercialization process of genetically modified breeding has been slow.

Although rice breeding ranks first globally when compared to other crops, the primary goals of conventional rice breeding focus on agronomic traits, such as high yield, high quality (mainly quality), as well as disease and insect resistance. Relatively little research has been conducted on functional nutritional rice in developing countries or research is at the preliminary stages when compared to that in developed countries. Presently, the major challenges associated with functional nutritional rice include the following. Rice breeding research has made three breakthroughs in germplasm resource exploration and utilization. Although there are over 100,000 global rice germplasm resources, those related to functional - nutritional rice are scarce. And the difficulty of cross - country germplasm exchange hinders the rapid breeding of such rice. Second, the molecular basis of research on functional nutritional rice is limited. The identification of genes related to functional nutritional rice is difficult, and the synthesis, metabolic pathways, and molecular regulatory mechanisms of biologically active substances are complex. Furthermore, methods of identifying and analyzing nutritional functional substances are lacking, thereby resulting in high development costs. Third, awareness and acceptance of functional nutritional rice by the public are generally low. The public is not well-informed on functional nutritional rice and they tend to think that it is GM rice. Functional nutritional rice does not have various names, such as functional rice, nutritious rice, selenium-rich rice, which makes it difficult for the public to understand. Although there have been several achievements in rice research, cultivars, and production technologies, the industrialization of nutritional rice is still at an initial stage with small-scale planting and limited market-related products. Influential and widely recognized functional nutritional rice brands are scarce, and key links in the industry chain and value chain are missing. Fourth, the national or industry standards for functional rice need to be improved. To date, no national or industry standards for functional rice and its products have been established, which affects the development of the functional rice industry. Therefore, it is imperative to establish standards, production technology processes, quality evaluation specifications, and clarify relevant labeling for functional nutritional rice.

Based on the current progresses of functional nutritional rice, future research should focus on the following aspects. First, strengthening the development and utilization of functional nutritional rice germplasm. A method for identifying functional nutritional rice germplasm from the existing rice germplasm resource library should be established, germplasm with unique biologically active substances should be extensively explored, developed, and utilized. Second, breeding of new functional nutritional rice varieties. Conventional breeding methods should be combined with modern breeding techniques, and molecular design breeding to accelerate the cultivation of new nutritional functional rice varieties. Third, strengthen basic research on nutritional functional rice. Key genes that regulate the metabolic pathways associated with the synthesis of biologically active substances should be explored, and their regulatory mechanisms and metabolic networks analyzed. Effective connection between basic research and breeding applications of nutritional functional rice should be promoted.

In future, the priorities for improving functional nutritional rice should focus on enhancing yield, optimizing plant type, and balancing taste. Understanding regulatory networks and metabolic pathways can lead to more precise and efficient breeding strategies. Due to the low yield and poor taste of commercially available rice varieties, the recognitions of growers and consumers are low. Simultaneously enhancing stress resistance to ensure stable yield and quality under adverse conditions. Strengthen micronutrients, especially iron, zinc and other microelements, to address global micronutrient deficiencies and improve the nutritional status of the population. The cultivation of rice varieties with specific health functions suitable for specific populations should also be a priority research direction.