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As one of the fastest-growing global economies in the world India faces contradiction with having an estimated 14% of the population as undernourished. Furthermore, the malnutrition status and immunity of vulnerable communities in India has been diminished due to COVID-19 pandemic. India is self-sufficient in food grain production. Policy approaches that tackle hunger through supplements, food provision via public distribution systems. The rate of improvement in nutritional status among a large section of the population has not kept pace with India’s significant gains in economic prosperity as well as increasing productivity during recent decades in spite of governmental plan to tackle hunger. Therefore, policy support has been gradually sifting from eradication of hunger to diminution of hidden hunger. Biofortification is one of the cost effective and environmentally and socially acceptable and ecologically friendly approach to enhance micronutrients, protein, vitamins and other important nutrition traits in edible food crops. Rice is the staple food of more than half of the Indian population. Therefore, biofortification in rice has substantial potentiality to enhance the nutritional status of the rice consumers. The Government of India has already taken an important step by providing policy and research support in linking agriculture and nutrition with biofortification. Prime Minister Narendra Modi has given a strong advocacy to staple crop biofortification as a natural and cost-effective solution to lessen malnutrition. On World Food Day in 2020, the Hon’ble Prime Minister dedicated to the nation 17 recently-developed biofortified varieties of eight indigenous and traditional crops, including rice, lacking key micronutrients that are essential for good health. He said, “We are taking an important step in our fight against malnutrition as we are now encouraging the production of crops which are rich in nutritional substances like protein, iron, zinc etc.”.

Introduction Malnutrition remains a pervasive global challenge, affecting millions of individuals across the world, particularly in low- and middle-income countries. Despite significant advancements in agriculture and food production, a large segment of the population still lacks access to essential nutrients, leading to severe health consequences and perpetuating cycles of poverty and underdevelopment (Müller and Krawinkel, 2005). Malnutrition, defined as the inadequate intake of essential nutrients required for optimal health and development, encompasses both undernutrition and over-nutrition (WHO, 2023). While undernutrition, characterized by deficiencies in energy, protein, vitamins, and minerals, remains a significant concern, over nutrition, manifesting as obesity and diet-related non-communicable diseases, is also on the rise, particularly in urban settings (Wells et al., 2020). The consequences of malnutrition are far-reaching, impacting physical growth, cognitive development, immune function, and overall well-being, with long term implications for productivity, economic prosperity, and societal stability (Saunders and Smith, 2010). Rice, a staple food for more than half of the world’s population, plays a central role in global food security and nutrition. However, conventional rice varieties often lack sufficient levels of key nutrients, such as iron, zinc, vitamin A, and certain amino acids, leaving populations reliant on rice-based diets vulnerable to micronutrient deficiencies and associated health risks (Peña-Rosas and Mithra, 2019). Iron deficiency anaemia, zinc deficiency, and vitamin A deficiency, are among the most prevalent forms of malnutrition worldwide, particularly affecting women and children in resource-constrained settings where rice consumption is high (Dipti et al., 2012)

Introduction: N metabolism Nitrogen is one of the three essential macronutrients for plant growth and yield (Su et al., 2005) and most crucial elements for root growth also (Fageria, 2014). Most macromolecules and a variety of secondary and signalling substances, such as proteins, nucleic acids, cell wall components, hormones, and vitamins, contain nitrogen (N), an important element. In order to assimilate nitrogen, nitrate must be reduced to ammonium, and then ammonium must be converted into amino acids. Perez et al. (1973) pointed out that the rice genotype with high protein yield translocated N from leaf blade to the developing grains more efficiently than the low protein genotype. This was reported due to a high concentration of free amino acids in the sap translocated to the developing grains rather than to differences in translocation rates. Therefore, proper understanding of N metabolism and amino acid translocation are prerequisite to comprehend higher grain protein in high yielding rice genotypes. Use of N by plants involves several steps including uptake, assimilation, translocation and remobilization; and recycling and remobilization when the plant is ageing (Fig. 1). The enzyme nitrate reductase (NR) assimilates nitrate, the main form of nitrogen available to agricultural plants in the field. This enzyme and the nitrogen status of various higher plant systems have a strong positive link, and growth, yield, or protein content can occasionally be connected to this enzyme’s level in seeds or leaves. Nitrate intake occurs at the root level, and it has been demonstrated that plants have two nitrate transport systems that work together to cohabit, absorb, and distribute nitrate from the soil solution throughout the entire plant. (Srivastava 1980) Following NO3- uptake by NO3- transporters and reduction of NO3- to NO2- by nitrate reductase (NR) in the cytoplasm, the process of NO3- assimilation commences (Lea and Miflin, 1974).

Introduction Understanding the variation in grain protein content is the pre-requisite of its improvement in modern rice varieties. The measure of quantitative and qualitative aspects of grain storage protein content includes estimation of soluble and total grain protein, quantifying various fractions, viz. glutelins, prolamins, globulins and albumins and their profiling through SDS-Page and  estimation of amino acid. As traditional chemical methods for protein content determination are destructive in nature. All these estimates comprehensively give insight into the diversity of storage protein in rice germplasm. The standard procedures followed for all these estimates are briefly stated in the  following sections. Estimation of Total Grain Protein Content in Rice The total protein is estimated via estimation of total N through the Kjeldahl method whereas soluble protein is estimated through Lowry method, Dumas method and Bradford method. But for estimation of large number of genotypes all these processes are tedious, time-consuming, and costly. Near-infrared spectroscopy (NIRS), as a non-destructive detection technique, can achieve rapid quantitative determination of protein contents and become an important development direction to replace traditional chemical detection methods.

Introduction Rice is one of the most important staple foods among all cereals. The larger population in Asia and Africa depends on rice for their daily calorie and nutritional requirements. People are found to bank on rice as their main source of nutrition in emerging and underdeveloped nations (Yang et al. 2016). However, it is considered that the milled or polished rice is nutritionally poor as the majority of the essential micronutrients viz. iron (Fe) and zinc (Zn), and important vitamins are lost during the process of milling and polishing (Johnson et al. 2011). So, by the way, the poorer section of the worldwide population mainly depends on rice and is most vulnerable to ‘hidden hunger’ as they are unable to afford other micronutrients-rich non-staple foods for their balanced diet and are often at the maximum risk for micronutrients deficiencies (Bouis and Saltzman 2017). The only possible solution for such malnutrition is to have nutritionally enriched food in the daily foodstuff. However, approximately one third of the world’s population is facing the problem of hidden hunger (White and Broadley 2009). Prolonged utilization of carbohydrate-rich food mainly based on rice, wheat, or maize is contributing to such nutritional deficiency in the poorer section of our society as most of them are unable to supplement nutritionally rich food for their diet. The deficiency of micronutrients can be termed a silent epidemic because it slowly weakens our immune system and hampers physical and intellectual development which may lead to fatality in some severe conditions. In the case of micronutrient deficiencies, deficiencies  of iron or iron deficiency anaemia (IDA), zinc deficiency, and vitamin-A  deficiency (VAD) are common, and have serious consequences.

Introduction Rice contains essential micronutrients such as iron (Fe), zinc (Zn), copper (Cu), calcium (Ca), manganese (Mn), magnesium (Mg), etc. which are generally lower when compared with other staple crops like wheat, maize, legumes, and tubers (Adeyeye et al. 2000). The most crucial micronutrients are iron and zinc, whose inadequacy is a leading cause of malnutrition. In this chapter we discuss estimation methodologies and diversity of Fe and Zn, its genetic basis, and improvement for micronutrient content through breeding techniques. Procedure of Estimation of Fe and Zn Content in Rice Grain Iron and zinc concentrations in rice samples were generally estimated by colorimetric method Atomic Absorption Spectrometry (AAS), X-ray Fluorescence (XRF), and Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES). X-Ray Fluorescence (XRF) Spectrometry is extremely helpful in the non-destructive estimation of relative Zn and Fe concentration in rice breeding lines and large germplasm collection (Paltridge et al. 2012). The majority of biofortification programs employ XRF as a metal analysis tool. For estimation of Fe and Zn, panicles of middle row plants of each replication were harvested, sun-dried for 4-5 days to reduce the moisture content to 11-12 %, and then seeds were dehusked. Each brown rice sample of about 5g was subjected to energy dispersive X-ray fluorescent spectrophotometer (ED-XRF) as per standard protocol (Stangoulis and Sison 2008, Rao et al. 2014) and expressed in ppm (µg g-1).

Introduction Rice is the world’s most important staple crop and is a primary source of energy and nutrition for about half of the world’s population. So it is the most important cereal crop with respect to food security of common people. Due to increasing world population and shrinkage of farm land holdings there is an urgent need to increase the grain productivity. Therefore, increasing grain yield has become the primary objective in many rice-breeding programs. India is the second highest rice producer (about 120 million ton/year) in the world and able to meet the demand of rice for the entire Indian population. But along with yield improvement of rice, quality has now become a foremost consideration for rice consumers and millers. Quality is defined as “the totality of features and characteristics of a product or service that bears its ability to satisfy stated or implied needs” (International Standard Organization (ISO) 8402 1986). The concept of grain quality covers many physico-chemical properties such as grain shape and size, milling and cooking properties, amylose content, stickiness, chalkiness, texture, colour, gelatinization characters and various nutritional properties. Thus, rice grain quality generally includes five main classes i.e. (i) milling quality, (ii) physical quality, (iii) cooking and eating quality (iv) sensory quality and (v) nutritional quality. Grain quality and its assessment are very important to consumers, millers and rice breeders who are engaged in varietal improvement programme, related to new features such as high nutritional quality, high yield potential, highly resistant to abiotic or biotic stresses, higher water use efficiency etc.

Introduction The transgenic approach can be an effective alternative for the development of biofortified crops when there is limited genetic variation for the targeted nutrient content among the germplasm (Zhu et al. 2007). Transgenic approaches have been used for the simultaneous integration of genes involved in the enhancement of micronutrient concentration as well as reduction in the concentration of antinutrients, resulting in enhancing the bioavailability of nutrients in plants. Development of biofortified crops employing the transgenic approaches, involves a significant amount of time, effort, and resources during the development stage, but it is a cost-effective and sustainable approach in a long run (Garg et al., 2018). For its wide consumption, rice has been targeted to address the global challenge of malnutrition. Vitamin deficiency can be considered as one of the significant challenges that affect poor section of populations due to low affordability. Development of ‘Golden Rice’ with provitamin A (beta-carotene) was recognized as an important breakthrough in this direction by expressing genes encoding PSY and carotene desaturase (Datta et al. 2003). Folic acid (vitamin B9) is important during pregnancy and reducing anemia. Rice was genetically modified to increase folate content (up to 150-fold) by overexpressing genes encoding Arabidopsis GTP-cyclohydrolase I (GTPCHI) and aminodeoxy-chorismate synthase (ADCS) (Blancquaert et al. 2015). Rice has also been targeted to address the global challenge of iron deficiency anemia. Multiple reports indicated an enhance in iron content by overexpressing a number of genes (Lee et al. 2009). Similarly, zinc content was also raised in rice by overexpressing OsIRT1 and mugineic acid biosynthesis genes from barley (HvNAS1, HvNAS1, HvNAAT-A, HvNAAT-B, IDS3) (Lee and An 2009, Masuda et al. 2008).

Significant Progress and Prospects It is widely acknowledged that biofortification presents a promising and cost effective agricultural strategy to enhance the nutritional status of malnourished populations globally. Approaches to biofortification, including crop breeding, targeted genetic modification, and mineral fertilizer application, offer significant potential for combatting mineral deficiencies in human diets. The development of biofortified food crops with enhanced nutrient content such as iron, zinc, selenium, and provitamin A has shown promise in addressing micronutrient deficiencies prevalent in both developing and developed nations. Over the past decade, both national and international initiatives have made substantial contributions toward achieving these goals. These efforts have resulted in the release of biofortified varieties across various crops, including rice, maize, wheat, potatoes, vegetables, and millets. The majority of these varieties have been developed through conventional breeding methods without sacrificing crop yield, making them readily accepted by both growers and consumers. One of the United Nations’ sustainable development goals is the cultivation of nutritionally rich crop varieties with heightened levels of micronutrients such as iron, zinc, calcium, total protein, lysine, tryptophan, anthocyanin, provitamin A, and oleic acid, coupled with reduced levels of anti-nutritional factors. In the past decade, 142 biofortified varieties, spanning rice, wheat, maize, pearl millet, small millet, lentil, chickpea, and other crops, have been developed under the auspices of the Indian Council of Agricultural Research (ICAR). Biofortified varieties typically do not impact ecological conditions, soil, or water requirements differently from traditional varieties. Additionally, they do not incur extra cultivation costs, and their economic output is comparable to traditional produce, leading to their widespread adoption. In India, the scale-up of biofortified varieties has gained momentum, with substantial quantities of breeder seeds being produced and distributed to public and private seed agencies for further multiplication and dissemination to farmers. Over the past six years, approximately 10 million hectares of land have been cultivated with biofortified rice, wheat, pearl millet, mustard, and lentil varieties.