A comprehensive review on mutation breeding milestones in cereals: Conventional to advanced molecular approaches to achieve sustainable goals in trait improvement

The grass family includes the annual common grasses known as cereals. The 5 main types of cereals are rice, wheat, barley, rye and oats. As a result of their high vitamin (20 - 80%) and fiber content (90%), these grains can help treat conditions like type II diabetes, complex metabolic syndrome, obesity, gastrointestinal disorders


Introduction
The word "cereal" is derived from the Latin word "cerealis," which means "grain" and refers to a specific kind of fruit called a caryopsis, which is made up of the endosperm, germ and bran (1).The world's population relies heavily on cereals as a source of energy, protein, B vitamins and minerals.They are the most significant food sources (2).Cereal grains were the earliest agricultural initiatives by early man and people still consume them now based on their location as well as what grows there best.Cereal grains are grown in huge amounts and produce more food energy globally than any other crop; therefore, they are staple food crops (1).The consumption of the major cereals is used as a treatment for various human diseases (3).Moreover, the prediction for global grain consumption has climbed by 2735 million tons in 2020, compared to 43 million tons (1.6%) in 2019-20 (4).The profound importance of cereals in economic and scientific realms has sparked extensive research on their genetics, development, and evolution, leading to a rich historical exploration.The almost full rice genome sequence serves as a symbol of the shift to highthroughput genomics and computational biology, which has influenced research on many kinds of cereals (5).

Many genetic variations have been created since
Muller and Stadler's groundbreaking work eighty years ago.These variations have been made possible by using a wide range of physical and chemical agents in mutation procedures.These procedures are now essential for plant breeding and genetic studies (6).Mutations are heritable changes in the phenotype of an organism.Chemical changes at the gene level are what cause these changes (7).Mutagens such as fast neutrons, ion beam radiation, Xrays, Gamma rays, and Ethyl methane sulphonate (EMS) are frequently used in mutation breeding.The physical mutation tends to result in double-strand DNA breaks and significant DNA deletions and consequently produces visible impacts on the chromosomal aberrations.Gamma rays are the most popular physical mutagen among plant breeders because they are convenient and may penetrate deeper into the tissues (8).Chemical mutagens like ethyl methane sulphonate and N-methyl-N-nitrosourea are widely used (9).It has most frequently been used to predominantly induce single-point mutation employed in investigations on reverse genetics.Mutation breeding is focused on taking advantage of local cultivars (10,11).The number of mutant varieties developed so far by physical and chemical mutagens is 2610 and 384 respectively (12).Induced mutations are utilized to produce mutant plant cultivars with altered plant traits, leading to a notable increase in production and improvement in quality.The generated mutants have helped plant breeders overcome many challenges and create new, useful forms, such as semi-dwarf varieties, early maturing, and disease-resistant varieties (13).For many agricultural plants, dwarfism is a favorable trait.The development of dwarf rice and wheat cultivars was a key component in the success of the Green Revolution.Dwarfism can minimize lodging and boost the harvest index in grain crops (14).Mutations in the genes governing the GA signaling pathway or its production frequently result in dwarfism.Over the past 50 years, mutation breeding has gained popularity and as of today, 3362 mutant plant types from 240 different plant species have been introduced in more than 75 nations among them 1602 major kinds of cereal, 501 major legumes, and 86 major oil seed mutant varieties are developed by mutation breeding/induced mutation (12).The review highlights the mutants discovered in the major cereals, their traits and genes the functional validation of mutants, and the application of the mutants in the breeding program to enhance the genetic gain of the cereal crop species (Fig. 1).

Rice:
Rice is the base of the global population's diet about 3.5 billion consume it around the world (15), hence the advancement of the rice sector, particularly in breeding, is essential (16).The limited genetic diversity of rice has also slowed the creation of cultivars resistant to biotic (37.4%) (17) and abiotic (50%-70%) (18) stresses, leading to yield loss (19).One of the finest methods for introducing variations among the mega types with good adaptation is to use induced mutations.To cause mutations in rice, physical agents such as fast neutron, gamma-ray, and ion beams as well as chemical mutagens like ethyl methane sulfonate (EMS), methyl nitrosourea (MNU), and sodium azide (SA) have been used (20).The major unfavourable traits of rice are tall, lodging, and susceptibility to various abiotic and biotic stresses.Semi-dwarfism is one of the most crucial features in many crops, including rice, and was made possible by the Green Revolution in 1966.The "green revolution gene" is commonly referred to as the rice semi-dwarf 1 (sd-1) gene.Sd-1, which was produced from the Chinese cultivar Dee-Geo-Woo-Gen (DGWG), gives rice cultivars short, thick culms, increases the harvest index, enhances lodging resistance, and improves sensitivity to nitrogenous fertilizers, producing large yields without degrading panicle and grain quality (21).Mutations in the genes regulating the production or signaling pathway of the plant hormone GA frequently result in dwarfism (22).According to recent research, 85 genes and at least 76 plant-height mutants have been found in rice (23).In India, several rice cultivars from the "PNR" series were introduced.A few of these cultivars grow quickly and have short plant stature.Two early ripening, aromatic mutation-derived rice varieties namely, PNR-381 and PNR-102, are being preferred by farmers in the states of Haryana and Uttar Pradesh (24), these are the main contributions to crop improvement.The identified elite mutants for various characters in rice are listed (Table 1).

Barley
Barley is a fast-growing plant that can survive in a variety of environments.Ancient Egypt and the Roman Empire cultivated cereals as early as 15,000 BC (3).Barley grows well in cold climates and contains less gluten and high fiber.Additionally, the nutrient status of barley includes minerals like sodium, potassium, calcium, magnesium, iron, zinc, and selenium, proteins, and vitamin B. Cultivated barley (Hordeum vulgare), is primarily farmed for animal feed, particularly for pigs and for malting and making beer, and for distilling whisky.Barley is utilized as a minor portion of the diet.In the UK and the Far and Middle East, pearled barley is consumed in soups and stews.In some nations, barley is also mashed into porridge and used in baking as flour (25).The discovery of the first disease-resistant mutant against "barley powdery mildew" dates back to 1942, marking the beginning of mutation breeding in barley.This achievement was made possible through the application of X-ray-induced mutagenesis (6) and the first, chemically induced mutant 'Luther' in barley was released in 1966 in the USA which gave 20% higher yield and lodging resistance (10).Nilsson-Ehle crossed erectoides mutants with the Danish Kenia variety, superior malting barley, and obtained lodging-resistant and highyielding recombinants (26).The major traits of barley improvement are semidwarf variety, lodging, good malting quality, and biotic and abiotic stress resistance (Table 2).

Wheat
Agronomically significant features have been successfully bred in a variety of crops using mutations.By using several mutagens, induced mutations in wheat have been discovered for both morphological and quantitative features (27).India's wheat production has grown rapidly to 96 million tonnes ever since the Green Revolution (28).However, the current breeding programs cannot increase production because they are not making the best use of genetic variety through recombination and selection.This circumstance demands the use of induced mutagenesis to produce fresh genetic variety.The major traits that should be improved in wheat are rust resistance, drought tolerance, short stature, early maturity, herbicide tolerance, and pre-harvest sprouting tolerance (Table 3).The most widely used genes in modern plant breeding are the semi-dwarf genes found in rice and wheat, which helped to spark a "green revolution" in the 1960s.The genes associated with the Green Revolution, namely RhtB1b and Rht-D1b, renowned for their capacity to reduce height, have been identified in over 95% of the wheat cultivars that have been introduced.In regions where the wheat crop is subject to terminal heat stress, early maturity is a desired characteristic.The flowering process in wheat is controlled by three gene sets: the photoperiod genes (Ppd), vernalization genes (Vrn), and earliness per se genes (eps), Early mutants were identified and confirmed in the M3 generation of 2 cultivars, PBW677 and HD2967 (29).

Oat
A significant cereal grain grown all over the world is oat (Avena sativa L.).After maize, rice, wheat, barley, and sorghum, oat output is sixth in the world (30).The oat crop has previously been neglected in several ways and grown in places that are not ideal for growing wheat, barley, or maize.The popularity of oats has grown recently because oat grains are rich in unique galactolipids, unsaturated fatty acids, avenanthramides, and important dietary minerals including iron.Due to its grain's superior nutritional composition, which includes high-quality proteins, dietary fibers, carbohydrates, oil, minerals, and low starch content, oats are typically utilized as highenergy feed (31).Oats constitute a substantial source of high-quality proteins, with approximately 12% to 15% of cultivated oats being rich in protein.Oats with even greater protein levels would be a fantastic source of protein from the plant source.230 mutant families with seed protein levels of 15% or greater, from a mutagenized oat population, were found in plants grown in greenhouses, 15 mutant lines with protein levels between 17 and 24% were chosen for future research (32).The identified elite mutants for various characteristics of oats are listed (Table 4).

Rye:
After wheat, rye (Secale cereale L.) is the second most employed grain for crafting bread.Rye can be grown in regions that are often unsuitable for other cereal crops because it is extremely winter-resistant and can survive in sandy soils with little fertility.Mostly, the rye planted worldwide is in cold temperate zones, but it may also be grown in semiarid areas close to deserts and at high altitudes (33).It is a significant crop in the Scandinavian nations, Germany, Poland, and Russia, where it is the main bread grain.Additionally, rye is utilized as animal feed and to make crispbread and alcohol.Genomics techniques were only recently created because Rye wasn't well known around the world and it was hard to genetically test a cross -pollinator with a genetic self-incompatibility mechanism (34).The identified elite mutants for various characters in Rye are listed (Table 5).

Integrative approaches in Mutation Breeding:
Mutation breeding involves 3 forms of mutagenesis.These are induced mutagenesis, in which mutations occur as a result of irradiation (gamma rays, X-rays, ion beam, etc.) or treatment with chemical mutagens; site-directed mutagenesis, which is the process of creating a mutation at a defined site in a DNA molecule and insertion mutagenesis, which is due to DNA insertions, either through genetic transformation and insertion of T-DNA or activation of transposable elements (35).Mutagenic plants or mutagenic seeds are developed and can be utilized directly as a commercial cultivar or as a parent to breed new commercial cultivars (36).Advanced mutation breeding methods and their outcomes are explained in (Fig 2).The M2 and M3 mutant populations can be used to map the altered genes using MutMap, MutMap + , and MutMap Gap.The genes identified can be utilized in functional validation and characterization.The elite mutant identified and developed has been used for multiomics research to study in-depth of the mutant.

Mutagenesis and Functional Validation:
Recently, the use of mutation breeding as a productive strategy for agricultural improvement has gained in popularity (6).The key outcomes of mutation breeding were quality traits like dwarfism, high yielding, early maturity, and tolerance to various biotic and abiotic stress; the resulting mutants are widely used for various purposes, such as improving both oligogenic and polygenic characters as well as morphological and physiological characters.They are employed to examine the genetic structure and provide functional validation.The direct use of mutations in the building of genetic maps in structural and functional genomics (Fig. 2) could quickly boost plant production and quality.The molecular methods for DNA fingerprinting and molecular mapping, including RAPD (Random Amplified Polymorphic DNA), AFLP (Amplified Fragment Length Polymorphisms), and STMS (Sequence-Tagged Microsatellite Sites), have made major contributions to the analysis and screening of mutants (6).Several rice genes have been functionally validated with the help of transgenics and EMS mutants (37).AKS-sd1, a derived cleaved amplified polymorphic sequence (dCAPS) marker, was created to validate the semi-dwarfing allele, sd1-bm (Bindli Mutant 34), This allele offers an option to the most used sd1-d in programs to improve rice (23).Two PCR markers were created to test for the presence of semi-dwarfing alleles in wheat the Rht-B1c and Rht-B1e.For Rht-B1c, a 256-bp product was amplified by PCR using the primer pair Rht-B1c-F1/Rht-B1c -R1 exclusively from lines bearing the Rht-B1c allele.For Rht-B1e, 2 primer sets were used: PCR with forward primer BF and reverse primer MR3 amplified a 228-bp fragment only in lines containing the Rht-B1e allele, while PCR with forward primer BF and reverse primer WR3 amplified wildtype sequences (38).

Application of Identified Mutant in Crop Improvement
Due to the limited amount of arable land present, the diminishing supply of water, and the fluctuating climatic conditions, the future of food production is gloomy.A decrease in arable land as a result of urbanization, salinization, biotic stress, drought, and desertification added to the issues.There are various methods for utilizing the heritable variations that are genetically encoded in existing crop plants for crop improvement programs (39).Such genetic alterations may take place experimentally under the influence of physical and chemical mutagens or may take place naturally at a very slow rate.Conventional mutation techniques have been employed frequently to enhance agricultural output, quality, disease, and insect resistance (Fig. 3) as well as the aesthetic appeal of flowers and ornamental plants.In most cases, mutation is the primary source of genetic variability, the raw material for natural selection-based evolution (40).

Conclusion
Over the last few years, mutation breeding has gained popularity and has been adopted by many countries to improve characters.It enhances various qualitative and quantitative crop plant characteristics and has been successfully employed in a variety of cereals, grain legumes, oil seeds, vegetables, fruits, medicinal plants, ornamental plants, and fodder crops.In this review, mutants identified in cereals were emphasized.These elite mutant varieties produced by mutation breeding can be used in hybridization programs for the introgression of superior genes in the different elite backgrounds using marker-assisted selection in the crop species.This will enhance the genetic gain by designing superior crop varieties.The next step in mutation breeding is multiomics studies, where the best mutants will be used for integrated omics research (genomics, transcriptomics, proteomics, metabolomics, and metagenomics) and functional characterization, as well as for creating superior crop varieties and enhancing crop quality.

Fig. 1 .
Fig. 1.Developmental milestones of desirable and beneficial mutants in cereal crops of rice, wheat, barley, oat, and rye.

Fig. 2 .Fig. 3 .
Fig. 2. Integrative approaches to mutation breeding and advanced multi-omic strategies to develop elite mutants with enhanced genetic gain.

Table 1 .
Identified elite mutants for various characters in rice

Table 2 .
Identified elite mutants for various characters in Barley

Table 3 .
Identified elite mutants for various characters in Wheat

Table 4 .
Identified elite mutants for various characters in Oat

Table 5 .
Identified elite mutants for various characters in Rye