Preface

Global food security is highly dependent on grain crops, which produce edible dry seeds that serve as a good source of protein, carbohydrates, and vitamins. Being the most critical component of a human diet, it is not astonishing that over 50% of world daily caloric intake is derived directly from grains. These crops are grown in greater quantities worldwide than any other crop and have undoubtedly played a key role in shaping human civilization.

Current crop production systems are under unprecedented pressure, not only due to the galloping world population but also because of changes in the climate. The frequent occurrence of extreme weather events such as drought and extreme temperatures are some of the key hindrances that threaten the sustainability of agricultural systems.

Recent developments in agriculture have only been possible due to the innovations and advancements in the field of biotechnology. Efforts are under way for development of molecular markers, mapping of genes, sequencing of marker genes, and their utilization through marker-assisted selection. Clustered, regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated 9 (Cas9) nucleases are some of the remarkable breakthroughs that are shaping the future's agriculture.

Ensuring proper water availability is pivotal for crop production. Scientists are focusing on developing drought resistant varieties of grain crops through conventional breeding methods as well as through the use of molecular biology and biotechnology.

Quality characteristics of grains are major determinants of market price, which includes milling, physical appearance, cooking, sensory, palatability, and nutritional value. A better understanding of the factors that regulate these quality characteristics will be useful for developing new breeding strategies.

Intensive production systems involving major grain crops all over the world have contributed to soil deterioration. One promissory approach is to emphasize soil sustainability through neglected and underutilized grain legumes (MNUGLs) to withstand the effects of abiotic stresses, climate change, and to boost nutritional security.

This book has been co-edited by editors from Pakistan and Turkey, and as a joint venture; we proudly dedicate this effort to the everlasting friendship between two brotherly nations: Pakistan and Turkey.

> **Farooq Shah** Abdul Wali Khan University Mardan, Pakistan

#### **Zafar Khan and Amjad Iqbal**

Abdul Wali Khan University, Pakistan

#### **Metin Turan**

Yeditepe University, Turkey

#### **Murat Olgun**

**1**

knowledge.

**Chapter 1**

*and Farooq Shah*

**1. Introduction**

Introductory Chapter: Recent

*Adil Hussain, Amjad Iqbal, Zafar Hayat Khan*

Advances in Grain Crops Research

Grain crops produce small, hard, dry seeds that may or may not have an attached

Among the grain crops, cereal grains occupied the central position concerning production, consumption, and health benefits. Cereal crops belong to the grass family, which is also known as Poaceae. The whole cereal grain consists of bran, endosperm, and the germ. Bran is the outermost layer having higher amounts of ɷ-3 fatty acids, fibers, macro and micro minerals, and vitamins. The endosperm mainly contains starch, where the germ consists of water and fat soluble vitamins, magnesium, and phosphorous. It is believed that the consumption of cereal grains has been co-started with human civilization that became the major part of human diet with the passage of time. Among the cereal grains, wheat, maize, rice, millet, buckwheat, and sorghum are used as a staple food by the people from different localities of the developed and under-developed world. It is assumed that about half of the daily caloric requirement is fulfilled through the consumption of cereal grains. A diet rich in cereals can in fact provide us with starches, soluble and insoluble fibers, digestible carbohydrates, essential fatty acids, appreciable amounts of

hull used for human and/or animal consumption. Grain crops are mainly of two different types, i.e., *cereals* and *legumes*. Grain crops are energy sources containing carbohydrates, proteins, and fats. Besides being the energy providing macromolecules, grain crops also contain health-supporting compounds, including dietary fibers, antioxidants, vitamins, and minerals [1]. The relatively higher durability of grain crops due to their low moisture content upon maturity has made them wellsuited to industrial agriculture as compared to other fleshy fruits and vegetables that have very low shelf life due to higher moisture content. It is these attributes of easy storage, measurements, and transportation that may well be the reasons for selection and domestication of the grain crops by our earliest ancestors. It is now widely accepted that the development of grain agriculture is one of the most important factors in permanent settlements of human ancestors and their divisions into different social classes in history [2]. Earliest evidence of plant domestication comes from the middle east where rye grains with domesticated traits have been recovered [3], whereas the first evidence of cereal crop domestication comes from 9000 BCE. Later on, continued domestication and the resultant shift from a *huntergatherer* society to a *settled agricultural* society continued for thousands of years mainly consisting of grain crops. However, this domestication was primarily based on intermittent trial and error driven by a crude knowledge of selection and choice. Later, the selection and use of grain crops research were based on more scientific

Eskişehir Osmangazi University, Turkey

#### **Chapter 1**

## Introductory Chapter: Recent Advances in Grain Crops Research

*Adil Hussain, Amjad Iqbal, Zafar Hayat Khan and Farooq Shah*

#### **1. Introduction**

Grain crops produce small, hard, dry seeds that may or may not have an attached hull used for human and/or animal consumption. Grain crops are mainly of two different types, i.e., *cereals* and *legumes*. Grain crops are energy sources containing carbohydrates, proteins, and fats. Besides being the energy providing macromolecules, grain crops also contain health-supporting compounds, including dietary fibers, antioxidants, vitamins, and minerals [1]. The relatively higher durability of grain crops due to their low moisture content upon maturity has made them wellsuited to industrial agriculture as compared to other fleshy fruits and vegetables that have very low shelf life due to higher moisture content. It is these attributes of easy storage, measurements, and transportation that may well be the reasons for selection and domestication of the grain crops by our earliest ancestors. It is now widely accepted that the development of grain agriculture is one of the most important factors in permanent settlements of human ancestors and their divisions into different social classes in history [2]. Earliest evidence of plant domestication comes from the middle east where rye grains with domesticated traits have been recovered [3], whereas the first evidence of cereal crop domestication comes from 9000 BCE. Later on, continued domestication and the resultant shift from a *huntergatherer* society to a *settled agricultural* society continued for thousands of years mainly consisting of grain crops. However, this domestication was primarily based on intermittent trial and error driven by a crude knowledge of selection and choice. Later, the selection and use of grain crops research were based on more scientific knowledge.

Among the grain crops, cereal grains occupied the central position concerning production, consumption, and health benefits. Cereal crops belong to the grass family, which is also known as Poaceae. The whole cereal grain consists of bran, endosperm, and the germ. Bran is the outermost layer having higher amounts of ɷ-3 fatty acids, fibers, macro and micro minerals, and vitamins. The endosperm mainly contains starch, where the germ consists of water and fat soluble vitamins, magnesium, and phosphorous. It is believed that the consumption of cereal grains has been co-started with human civilization that became the major part of human diet with the passage of time. Among the cereal grains, wheat, maize, rice, millet, buckwheat, and sorghum are used as a staple food by the people from different localities of the developed and under-developed world. It is assumed that about half of the daily caloric requirement is fulfilled through the consumption of cereal grains. A diet rich in cereals can in fact provide us with starches, soluble and insoluble fibers, digestible carbohydrates, essential fatty acids, appreciable amounts of

proteins, higher amounts of folate, iron, magnesium, copper, phosphorus, zinc, and phytochemicals. The phytochemicals with antioxidant properties can lower the blood cholesterol levels and thus help in controlling heart diseases. In short, the whole cereal grain consists of a whole range of primary and secondary metabolites that not only provides energy, but also health supporting substances [4]. The phytochemicals or secondary metabolites of whole cereal grains are also known to be associated with significant health benefits. The phytochemicals that are rich in cereal grains include phenolics, lignans, phytic acid, phytosterols, saponin, squalene, tocotrienols, phytosterol, and oryzanol. Some of these phytochemicals can act as antioxidants and lower the blood cholesterol level, thus preventing the cardiovascular diseases and reduces the risk of cancer. On the other hand, industrial revolution led to the milling of the cereal grains that intensively involve the removal of the bran and germ. Albeit milling has improved the sensory attributes of the finished product, but the milled products lack important health beneficiary components, such as dietary fibers, phenolics, minerals, and vitamins [4]. Carbohydrates are the main class of primary metabolites that attribute the health benefits of cereal grains. It is known for the years that insoluble polysaccharides/ fibers can relax constipation, whereas the soluble polysaccharides/mixed-link β-glucans can regulate the blood cholesterol level. The soluble and non-soluble polysaccharides that are present naturally in cereal grains have positive health implications in humans. These polysaccharides have the ability to hold water, give bulk to the stool, and can be broken down into short chain fatty acids. Such activities may help to resist the degenerative diseases, i.e., coronary heart diseases and cancer [5].

Another important group of grain crops include legumes that belong to the Leguminosae, which is also named as Fabaceae. Legumes are recognized as pulses after decortication or dehulling of seeds. Legumes are grown on a large area worldwide. The important grain legumes include chickpea, cowpea, lentil, green peas, etc. Pulses are cultivated in tropical and temperate regions around the globe, mainly in the South-East Asia. Food legumes are considered to be healthy plant-based food and are a good source of vitamins (B-complex), minerals (macro and microminerals), and dietary fiber [6]. Chickpea seed has high protein and carbohydrate with small amounts of fiber, oil, vitamins, and minerals. The digestibility of pulses proteins varies from 70–80%. Pulses have a hypocholesteremic effect, and the seeds are eaten fresh as green vegetables or boiled with condiments. The flour of some pulses, such as chickpea is used in food preparation as a dressing for vegetables (e.g., bringle and potato) and meat (e.g., fish and chicken). The flour of chickpea is utilized for making bread with pepper and salt.

Pulses are good sources of protein that add to the nutritional importance of cereal-based diets. Pulse proteins are abundant in amino acid, arginine and lysine, but they have lower amounts of cysteine and methionine, which are known as sulfur containing amino acids. The other limiting essential amino acids include the cyclic amino acid tryptophan, valine, and threonine. From the nutritional standpoint, it is necessary to provide the body with an appropriate amount of essential amino acids. To meet the human dietary needs the four most important essential amino acids of protein should be present in the ratio of: four parts of lysine, three parts of methionine, two parts of threonine, and one part of tryptophan. If any one of these amino acids is so low as to affect the above ratio, it becomes the limiting factor in determining the nutritive value of the protein. The content of the proteins in pulses is almost double as compared to the cereals. Moreover, pulses taken with cereals in diet can fulfill the basic requirements for protein and thus enhance the nutritional importance of cereal-based diets [7].

**3**

*Introductory Chapter: Recent Advances in Grain Crops Research*

Pulses are known to be good sources of macro and macro-minerals, mainly calcium (Ca), magnesium (Mg), potassium (K), phosphorus (P), zinc (Zn), and iron (Fe). These minerals are important for the body because of their role in physiological as well as biochemical processes of the cells and tissues. Mg is an integral part of metabolic enzymes, K helps in the regulation of energy and metabolism, P gives strength to the bone and is involved in the formation of high-energy compounds, i.e., AMP, ADP, and ATP. Likewise, Zn also acts as a cofactor of certain enzymes, and Fe is a part of hemoglobin and myoglobin. The ionized minerals can help in blood coagulation and keep the membrane permeability intact. Besides, sodium (Na) and K are vital for the regulation of an acid-base balance and osmotic pressure of the cells. Legumes also contain antinutrients, i.e., phytate, which is rich in P. Phytic acid (Inositol hexaphosphoric acid) is abundant in legumes that can inhibit the absorption of Zn and other essential elements by the small intestine of humans and monogastric animals, thus making it unavailable to the body. Phytic acid forms complexes with the metal ions (Cu+2, Ca+2, Zn+2, and Fe+3), thus excreting them out of the body. The excretion of such essential elements may lead to various deficiency diseases. Phytate may also limit the absorption of vitamins and

proteins, which can decrease the nutritional value of the legumes [8].

of the grain crop cultivars grown these days are indehiscent.

Other important aspects of research on grain crops include factors responsible for quantity and quality of the produce. Such factors have been studied by scientists throughout the world and in almost all grain crops. Most of these studies describe

Furthermore, several grain crops are also required as a source of animal feed. Recent interest in wheat research is supported by the directives following the meeting of the G20 ministries of agriculture, of the wheat initiative [9] to profess the coordination of international research on wheat to fulfill global wheat production demand. Global population is predicted to increase up to over 9 billion by 2050 [10], which means that the demand will increase by 60% as compared to that in 2010. In order to meet this end, global wheat production must increase by 1.6% annually, compared to the current 1% global increase per year (2001–2010) – reviewed by Giraldo et al. [11]. Other initiatives such as the International Barley Hub [12] started to support the increasing demand of grain crops. In this chapter, we discuss the recent advances made in grain crops research, related to few important traits. Seed shattering is a character that has probably played one of the most important roles in domestication of grain crops. Grain crop varieties or cultivars with less auto-shattering of the seeds, siliques, seed pods, and spikelets have been more desirable with regards to farming [13]. Evolutionary studies show that the loss of seeds shattering was a character independently acquired by monocots and dicots via "convergent phenotypic evolution" producing low dehiscent and/or indehiscent crop species. At tissue level, seed or seed pod shattering level is governed by histological modifications that involve the development of an abscission layer at the point where the seeds or spikelets/pods are attached with the plant [14–17]. Pourkheirandish et al. [18] demonstrated that a lower thickness of both primary and secondary cell walls of the abscission or separation layer results in higher seeds shattering in barley. Because of the importance that seed shattering carries in the adoption of grain crops for food and feed, it has been studied in detail in various grain crops at genetic level. A plethora of genes have been shown to regulate seed shattering in rice [19–22], barley [18], and legume crops such as soybean [23, 24], cow pea [25], common beans [26, 27], and medicago [28]. These genes encode various proteins such as the Shatterproof protein, NAC, Myc, and bHLH transcription factors, ALCATRAZ and the BEL-1-type homeobox SH5. Careful selection of crop varieties and cultivars by the farmers and scientific research have settled the seed shattering issue and most

*DOI: http://dx.doi.org/10.5772/intechopen.90701*

#### *Introductory Chapter: Recent Advances in Grain Crops Research DOI: http://dx.doi.org/10.5772/intechopen.90701*

*Recent Advances in Grain Crops Research*

proteins, higher amounts of folate, iron, magnesium, copper, phosphorus, zinc, and phytochemicals. The phytochemicals with antioxidant properties can lower the blood cholesterol levels and thus help in controlling heart diseases. In short, the whole cereal grain consists of a whole range of primary and secondary metabolites that not only provides energy, but also health supporting substances [4]. The phytochemicals or secondary metabolites of whole cereal grains are also known to be associated with significant health benefits. The phytochemicals that are rich in cereal grains include phenolics, lignans, phytic acid, phytosterols, saponin, squalene, tocotrienols, phytosterol, and oryzanol. Some of these phytochemicals can act as antioxidants and lower the blood cholesterol level, thus preventing the cardiovascular diseases and reduces the risk of cancer. On the other hand, industrial revolution led to the milling of the cereal grains that intensively involve the removal of the bran and germ. Albeit milling has improved the sensory attributes of the finished product, but the milled products lack important health beneficiary components, such as dietary fibers, phenolics, minerals, and vitamins [4]. Carbohydrates are the main class of primary metabolites that attribute the health benefits of cereal grains. It is known for the years that insoluble polysaccharides/ fibers can relax constipation, whereas the soluble polysaccharides/mixed-link β-glucans can regulate the blood cholesterol level. The soluble and non-soluble polysaccharides that are present naturally in cereal grains have positive health implications in humans. These polysaccharides have the ability to hold water, give bulk to the stool, and can be broken down into short chain fatty acids. Such activities may help to resist the degenerative diseases, i.e., coronary heart diseases and

Another important group of grain crops include legumes that belong to the Leguminosae, which is also named as Fabaceae. Legumes are recognized as pulses after decortication or dehulling of seeds. Legumes are grown on a large area worldwide. The important grain legumes include chickpea, cowpea, lentil, green peas, etc. Pulses are cultivated in tropical and temperate regions around the globe, mainly in the South-East Asia. Food legumes are considered to be healthy plant-based food and are a good source of vitamins (B-complex), minerals (macro and microminerals), and dietary fiber [6]. Chickpea seed has high protein and carbohydrate with small amounts of fiber, oil, vitamins, and minerals. The digestibility of pulses proteins varies from 70–80%. Pulses have a hypocholesteremic effect, and the seeds are eaten fresh as green vegetables or boiled with condiments. The flour of some pulses, such as chickpea is used in food preparation as a dressing for vegetables (e.g., bringle and potato) and meat (e.g., fish and chicken). The flour of chickpea is

Pulses are good sources of protein that add to the nutritional importance of cereal-based diets. Pulse proteins are abundant in amino acid, arginine and lysine, but they have lower amounts of cysteine and methionine, which are known as sulfur containing amino acids. The other limiting essential amino acids include the cyclic amino acid tryptophan, valine, and threonine. From the nutritional standpoint, it is necessary to provide the body with an appropriate amount of essential amino acids. To meet the human dietary needs the four most important essential amino acids of protein should be present in the ratio of: four parts of lysine, three parts of methionine, two parts of threonine, and one part of tryptophan. If any one of these amino acids is so low as to affect the above ratio, it becomes the limiting factor in determining the nutritive value of the protein. The content of the proteins in pulses is almost double as compared to the cereals. Moreover, pulses taken with cereals in diet can fulfill the basic requirements for protein and thus enhance the

utilized for making bread with pepper and salt.

nutritional importance of cereal-based diets [7].

**2**

cancer [5].

Pulses are known to be good sources of macro and macro-minerals, mainly calcium (Ca), magnesium (Mg), potassium (K), phosphorus (P), zinc (Zn), and iron (Fe). These minerals are important for the body because of their role in physiological as well as biochemical processes of the cells and tissues. Mg is an integral part of metabolic enzymes, K helps in the regulation of energy and metabolism, P gives strength to the bone and is involved in the formation of high-energy compounds, i.e., AMP, ADP, and ATP. Likewise, Zn also acts as a cofactor of certain enzymes, and Fe is a part of hemoglobin and myoglobin. The ionized minerals can help in blood coagulation and keep the membrane permeability intact. Besides, sodium (Na) and K are vital for the regulation of an acid-base balance and osmotic pressure of the cells. Legumes also contain antinutrients, i.e., phytate, which is rich in P. Phytic acid (Inositol hexaphosphoric acid) is abundant in legumes that can inhibit the absorption of Zn and other essential elements by the small intestine of humans and monogastric animals, thus making it unavailable to the body. Phytic acid forms complexes with the metal ions (Cu+2, Ca+2, Zn+2, and Fe+3), thus excreting them out of the body. The excretion of such essential elements may lead to various deficiency diseases. Phytate may also limit the absorption of vitamins and proteins, which can decrease the nutritional value of the legumes [8].

Furthermore, several grain crops are also required as a source of animal feed. Recent interest in wheat research is supported by the directives following the meeting of the G20 ministries of agriculture, of the wheat initiative [9] to profess the coordination of international research on wheat to fulfill global wheat production demand. Global population is predicted to increase up to over 9 billion by 2050 [10], which means that the demand will increase by 60% as compared to that in 2010. In order to meet this end, global wheat production must increase by 1.6% annually, compared to the current 1% global increase per year (2001–2010) – reviewed by Giraldo et al. [11]. Other initiatives such as the International Barley Hub [12] started to support the increasing demand of grain crops. In this chapter, we discuss the recent advances made in grain crops research, related to few important traits.

Seed shattering is a character that has probably played one of the most important roles in domestication of grain crops. Grain crop varieties or cultivars with less auto-shattering of the seeds, siliques, seed pods, and spikelets have been more desirable with regards to farming [13]. Evolutionary studies show that the loss of seeds shattering was a character independently acquired by monocots and dicots via "convergent phenotypic evolution" producing low dehiscent and/or indehiscent crop species. At tissue level, seed or seed pod shattering level is governed by histological modifications that involve the development of an abscission layer at the point where the seeds or spikelets/pods are attached with the plant [14–17]. Pourkheirandish et al. [18] demonstrated that a lower thickness of both primary and secondary cell walls of the abscission or separation layer results in higher seeds shattering in barley. Because of the importance that seed shattering carries in the adoption of grain crops for food and feed, it has been studied in detail in various grain crops at genetic level. A plethora of genes have been shown to regulate seed shattering in rice [19–22], barley [18], and legume crops such as soybean [23, 24], cow pea [25], common beans [26, 27], and medicago [28]. These genes encode various proteins such as the Shatterproof protein, NAC, Myc, and bHLH transcription factors, ALCATRAZ and the BEL-1-type homeobox SH5. Careful selection of crop varieties and cultivars by the farmers and scientific research have settled the seed shattering issue and most of the grain crop cultivars grown these days are indehiscent.

Other important aspects of research on grain crops include factors responsible for quantity and quality of the produce. Such factors have been studied by scientists throughout the world and in almost all grain crops. Most of these studies describe

both negative or positive effects of different interventions and treatments. Research targeted toward the understanding of genetic basis of crop improvement is often conducted under restricted or unfavorable environmental conditions. Such projects include research on uptake and use efficiency of various macro and micro nutrients [29, 30]. Chen et al. [31] identified at least 10 genes related to nitrogen metabolism in different cultivars of barley. Most interestingly, these included the 2nd isoform of nitrate reductase enzyme (HvNIA2), which is responsible for catalyzing the 1st reaction in nitrate assimilation, i.e., the conversion of nitrate to nitrite and is also involved in the production of nitric oxide (NO) in plants [32]. Other genes induced under low nitrogen stress included those encoding enzymes such as the chloroplastic glutamine synthetase (HvGS2), ferredoxin dependent glutamate synthase (HvGLU2), and asparagine synthetase (HvASN1). Interestingly, the expression of these genes was found to be strictly regulated by nitrogen stress and in a tissue specific manner [31].

The elucidation of the genetic role played by the members of the plant nitrate transporters (NRT) or peptide transporters (PTR) is also of particular importance in the transportation of various nutrients from the soil to plants and ultimately crop production. Lin et al. [33] in the year 2000 cloned and functionally characterized NRT1 from rice. The NRT is constitutively expressed in the epidermis and root hairs of the roots. The importance of this gene in nitrate transport can be determined by the fact that even slight variations in the sequence of this gene results in nitrate-use divergence in different rice subspecies [34]. This is associated with the higher nitrate-absorption activity of Indica rice as compared to Japonica rice. As the Japonica rice plants expressing the Indica NRT1.1B allele showed significantly higher grain yield and nitrogen-use efficiency compared to Japonica plants that did not express the Indica allele [34]. The role of plant NRT and their functional applications has been reviewed by Fan et al. [35]. Plant NRT/PTR gene family has been renamed as NPF family [36] and contains a large number of genes that can be subdivided into at about 10 sub-families. The family has 93 known genes in rice [37]. NPF members not only transport nitrate but a wide range of other substrates including peptides [38], phytohormones such as auxin [39], abscisic acid [40], Jasmonates [41], and Gibberellins [42] further indicating the importance of these genes in plant physiology and crop production. Several members of the family have remained a focus of different scientific studies, especially in rice, describing significant increase in the overall yield, early maturity and the composition of soil microbiota [43–45] and should be a topic of future research in grain crops.

After decades of research in different living organisms, it has now become clear that practical utilization of recent advances in modern biotechnology is inevitable. Plant science has probably benefited the most from biotechnological advancements in terms of modifications at genome and proteome levels. With the advent of different gene editing techniques such as the CRISPR/Cas technology, any type of genetic modification has become easy and quick. A simple search on the web of science using "CRISPR rice" as keywords turns up 96 different research papers published from 2013 to 2020. Similarly, a search for "CRISPR wheat" shows up 25 research papers between 2017 and 2019, whereas searching for "CRISPR maize" shows up 17 research papers. This indicates the rapid adoption of newly developed techniques by plant scientists for genetic modifications in these plants. The use CRISPR/ Cas technology for genetic modifications in grain crops has been described in the chapter by Hussain et al. [46] available as *online first* (https://www.intechopen.com/ online-first/crispr-cas9-mediated-gene-editing-in-grain-crops). In this scenario, the availability of genome sequences and other genetic resources of grain crops such as rice (Rice Genome Annotation Project http://rice.plantbiology.msu.edu/), wheat (International Wheat Genome Sequencing Consortium https://www.wheatgenome.

**5**

**Author details**

Khyber Pakhtunkhwa, Pakistan

provided the original work is properly cited.

Adil Hussain, Amjad Iqbal, Zafar Hayat Khan and Farooq Shah\* Department of Agriculture, Abdul Wali Khan University Mardan,

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Introductory Chapter: Recent Advances in Grain Crops Research*

org/), maize (Maize Genetics and Genomics Database https://www.maizegdb. org/), soybean (Integrating Genetics and Genomics to Advance Soybean Research: SoyBase https://www.soybase.org/), and other legumes (Legume Information system https://www.legumeinfo.org/) has further facilitated scientific research on grain crops at the cell or genome level. Further scientometric, informetric, and bibliometric studies are required to establish a detailed account for the impact of

*DOI: http://dx.doi.org/10.5772/intechopen.90701*

recent advances in grain crops research.

*Introductory Chapter: Recent Advances in Grain Crops Research DOI: http://dx.doi.org/10.5772/intechopen.90701*

*Recent Advances in Grain Crops Research*

specific manner [31].

both negative or positive effects of different interventions and treatments. Research targeted toward the understanding of genetic basis of crop improvement is often conducted under restricted or unfavorable environmental conditions. Such projects include research on uptake and use efficiency of various macro and micro nutrients [29, 30]. Chen et al. [31] identified at least 10 genes related to nitrogen metabolism in different cultivars of barley. Most interestingly, these included the 2nd isoform of nitrate reductase enzyme (HvNIA2), which is responsible for catalyzing the 1st reaction in nitrate assimilation, i.e., the conversion of nitrate to nitrite and is also involved in the production of nitric oxide (NO) in plants [32]. Other genes induced under low nitrogen stress included those encoding enzymes such as the chloroplastic glutamine synthetase (HvGS2), ferredoxin dependent glutamate synthase (HvGLU2), and asparagine synthetase (HvASN1). Interestingly, the expression of these genes was found to be strictly regulated by nitrogen stress and in a tissue

The elucidation of the genetic role played by the members of the plant nitrate transporters (NRT) or peptide transporters (PTR) is also of particular importance in the transportation of various nutrients from the soil to plants and ultimately crop production. Lin et al. [33] in the year 2000 cloned and functionally characterized NRT1 from rice. The NRT is constitutively expressed in the epidermis and root hairs of the roots. The importance of this gene in nitrate transport can be determined by the fact that even slight variations in the sequence of this gene results in nitrate-use divergence in different rice subspecies [34]. This is associated with the higher nitrate-absorption activity of Indica rice as compared to Japonica rice. As the Japonica rice plants expressing the Indica NRT1.1B allele showed significantly higher grain yield and nitrogen-use efficiency compared to Japonica plants that did not express the Indica allele [34]. The role of plant NRT and their functional applications has been reviewed by Fan et al. [35]. Plant NRT/PTR gene family has been renamed as NPF family [36] and contains a large number of genes that can be subdivided into at about 10 sub-families. The family has 93 known genes in rice [37]. NPF members not only transport nitrate but a wide range of other substrates including peptides [38], phytohormones such as auxin [39], abscisic acid [40], Jasmonates [41], and Gibberellins [42] further indicating the importance of these genes in plant physiology and crop production. Several members of the family have remained a focus of different scientific studies, especially in rice, describing significant increase in the overall yield, early maturity and the composition of soil

microbiota [43–45] and should be a topic of future research in grain crops.

After decades of research in different living organisms, it has now become clear that practical utilization of recent advances in modern biotechnology is inevitable. Plant science has probably benefited the most from biotechnological advancements in terms of modifications at genome and proteome levels. With the advent of different gene editing techniques such as the CRISPR/Cas technology, any type of genetic modification has become easy and quick. A simple search on the web of science using "CRISPR rice" as keywords turns up 96 different research papers published from 2013 to 2020. Similarly, a search for "CRISPR wheat" shows up 25 research papers between 2017 and 2019, whereas searching for "CRISPR maize" shows up 17 research papers. This indicates the rapid adoption of newly developed techniques by plant scientists for genetic modifications in these plants. The use CRISPR/ Cas technology for genetic modifications in grain crops has been described in the chapter by Hussain et al. [46] available as *online first* (https://www.intechopen.com/ online-first/crispr-cas9-mediated-gene-editing-in-grain-crops). In this scenario, the availability of genome sequences and other genetic resources of grain crops such as rice (Rice Genome Annotation Project http://rice.plantbiology.msu.edu/), wheat (International Wheat Genome Sequencing Consortium https://www.wheatgenome.

**4**

org/), maize (Maize Genetics and Genomics Database https://www.maizegdb. org/), soybean (Integrating Genetics and Genomics to Advance Soybean Research: SoyBase https://www.soybase.org/), and other legumes (Legume Information system https://www.legumeinfo.org/) has further facilitated scientific research on grain crops at the cell or genome level. Further scientometric, informetric, and bibliometric studies are required to establish a detailed account for the impact of recent advances in grain crops research.

### **Author details**

Adil Hussain, Amjad Iqbal, Zafar Hayat Khan and Farooq Shah\* Department of Agriculture, Abdul Wali Khan University Mardan, Khyber Pakhtunkhwa, Pakistan

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Belobrajdic DP, AR B. The potential role of phytochemicals in wholegrain cereals for the prevention of type-2 diabetes. Nutrition Journal. 2013;**12**(1):62

[2] Wessel TJA, Values H. The agricultural foundations of civilization. Agriculture Human Values. 1984;**1**(2):9-12

[3] Hillman G, Hedges R, Moore A, Colledge S, Pettitt P. New evidence of Lateglacial cereal cultivation at Abu Hureyra on the Euphrates. The Holocene. 2001;**11**(4):383-393

[4] Sarwar MH, Sarwar MF, Sarwar M, Qadri NA, Moghal S. The importance of cereals (Poaceae: Gramineae) nutrition in human health: A review. Journal of Cereals. 2013;**4**(3):32-35

[5] Kumar V, Sinha AK, Makkar HP, De Boeck G, Becker K. Dietary roles of non-starch polysaccharides in human nutrition: A review. Critical Reviews in Food Science. 2012;**52**(10):899-935

[6] Jukanti AK, Gaur PM, Gowda C, Chibbar RN. Nutritional quality and health benefits of chickpea (*Cicer arietinum* L.): A review. British Journal of Nutrition. 2012;**108**(S1):S11-S26

[7] Iqbal A, Khalil IA, Ateeq N, MS K. Nutritional quality of important food legumes. Food Chemistry. 2006;**97**(2):331-335

[8] Nkhata SG, Ayua E, Kamau EH, Shingiro JB. Fermentation and germination improve nutritional value of cereals and legumes through activation of endogenous enzymes. Food Science. 2018;**6**(8):2446-2458

[9] Wheat, Initiative. Available from: https://wwwwheatinitiativeorg/ [Accessed: November 2019]

[10] United Nations Department of Economics and Social Affairs. Population Division. Available from: https://www.un.org/en/development/ desa/population/index.asp [Accessed: November 2019]

[11] Giraldo P, Benavente E, Manzano-Agugliaro F, Gimenez EJA. Worldwide research trends on wheat and barley: A bibliometric comparative analysis. Agronomy. 2019;**9**(7):352

[12] International Barley Hub. Available from: http://www.barleyhub.org/ [Accessed: November 2019]

[13] Di Vittori V, Gioia T, Rodriguez M, Bellucci E, Bitocchi E, Nanni L, et al. Convergent evolution of the seed shattering trait. Genes (Basel). 2019;**10**(1):1-16

[14] Liljegren SJ, Ditta GS, Eshed Y, Savidge B, Bowman JL, Yanofsky MF. SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis. Nature. 2000;**404**(6779):766-770

[15] Rajani S, Sundaresan V. The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence. Current Biology. 2001;**11**(24):1914-1922

[16] Mitsuda N, Ohme-Takagi M. NAC transcription factors NST1 and NST3 regulate pod shattering in a partially redundant manner by promoting secondary wall formation after the establishment of tissue identity. The Plant Journal. 2008;**56**(5):768-778

[17] Mitsuda N, Iwase A, Yamamoto H, Yoshida M, Seki M, Shinozaki K, et al. NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell. 2007;**19**(1):270-280

**7**

*Introductory Chapter: Recent Advances in Grain Crops Research*

[26] Koinange EMK, Singh SP, Gepts P. Genetic control of the domestication syndrome in common bean. Crop Science. 1996;**36**:1037-1045

[27] Rau D, Murgia ML, Rodriguez M, Bitocchi E, Bellucci E, Fois D, et al. Genomic dissection of pod shattering in common bean: Mutations at non-orthologous loci at the basis of convergent phenotypic evolution under domestication of leguminous species. The Plant Journal. 2019;**97**(4):693-714

[28] Fourquin C, del Cerro C, Victoria FC, Vialette-Guiraud A, de Oliveira AC, Ferrándiz C. A change in SHATTERPROOF protein lies at the origin of a fruit morphological novelty and a new strategy for seed dispersal in Medicago genus. Plant Physiology.

[29] Cormier F, Foulkes J, Hirel B, Gouache D, Moënne-Loccoz Y, Le Gouis J. Breeding for increased nitrogen-use efficiency: A review for wheat (T. aestivum L.). Plant Breeding.

[30] Hu B, Wang W, Ou S, Tang J, Li H, Che R, et al. Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies. Nature Genetics. 2015 Jul;**47**(7):834-838

[31] Chen Z, Liu C, Wang Y, He T, Gao R, Xu H, et al. Expression analysis of nitrogen metabolism-related genes reveals differences in adaptation to lownitrogen stress between two different barley cultivars at seedling stage. International Journal of Genomics.

[32] Astier J, Gross I, Durner J. Nitric oxide production in plants: An update. Journal of Experimental Botany.

[33] Lin CM, Koh S, Stacey G, Yu SM, Lin TY, Tsay YF. Cloning and functional characterization of a constitutively

2013;**162**(2):907-917

2016;**135**(3):255-278

2018;**2018**:1-10

2018;**69**(14):3401-3411

*DOI: http://dx.doi.org/10.5772/intechopen.90701*

[18] Pourkheirandish M, Hensel G, Kilian B, Senthil N, Chen G, Sameri M, et al. Evolution of the grain dispersal system in barley. Cell. 2015;**162**(3):

[19] Konishi S, Izawa T, Lin SY, Ebana K, Fukuta Y, Sasaki T, et al. An SNP caused loss of seed shattering during rice domestication. Science.

2006;**312**(5778):1392-1396

[20] Li C, Zhou A, Sang T. Rice

domestication by reducing shattering. Science. 2006;**311**(5769):1936-1939

[21] Zhou Y, Lu D, Li C, Luo J, Zhu BF, Zhu J, et al. Genetic control of seed shattering in rice by the APETALA2 transcription factor shattering abortion.

Choi H, Koh HJ, An G. The BEL1-type homeobox gene SH5 induces seed shattering by enhancing abscissionzone development and inhibiting lignin biosynthesis. The Plant Journal. 2014

[23] Funatsuki H, Suzuki M, Hirose A, Inaba H, Yamada T, Hajika M, et al. Molecular basis of a shattering

resistance boosting global dissemination

of soybean. Proceedings of the National Academy of Sciences of the United States of America. 2014;**111**(50):17797-17802

[24] Dong Y, Yang X, Liu J,

Breeding. 2016;**36**(6):80

Wang B-H, Liu B-L, Wang Y-Z. Pod shattering resistance associated with domestication is mediated by a NAC gene in soybean. Nature Communications. 2014;**5**(1):3352

[25] Suanum W, Somta P, Kongjaimun A, Yimram T, Kaga A, Tomooka N, et al. Co-localization of QTLs for pod fiber content and pod shattering in F2 and backcross populations between yardlong bean and wild cowpea. Molecular

Plant Cell. 2012;**24**(3):1034-1048

[22] Yoon J, Cho LH, Kim SL,

Sep;**79**(5):717-728

527-539

#### *Introductory Chapter: Recent Advances in Grain Crops Research DOI: http://dx.doi.org/10.5772/intechopen.90701*

[18] Pourkheirandish M, Hensel G, Kilian B, Senthil N, Chen G, Sameri M, et al. Evolution of the grain dispersal system in barley. Cell. 2015;**162**(3): 527-539

[19] Konishi S, Izawa T, Lin SY, Ebana K, Fukuta Y, Sasaki T, et al. An SNP caused loss of seed shattering during rice domestication. Science. 2006;**312**(5778):1392-1396

[20] Li C, Zhou A, Sang T. Rice domestication by reducing shattering. Science. 2006;**311**(5769):1936-1939

[21] Zhou Y, Lu D, Li C, Luo J, Zhu BF, Zhu J, et al. Genetic control of seed shattering in rice by the APETALA2 transcription factor shattering abortion. Plant Cell. 2012;**24**(3):1034-1048

[22] Yoon J, Cho LH, Kim SL, Choi H, Koh HJ, An G. The BEL1-type homeobox gene SH5 induces seed shattering by enhancing abscissionzone development and inhibiting lignin biosynthesis. The Plant Journal. 2014 Sep;**79**(5):717-728

[23] Funatsuki H, Suzuki M, Hirose A, Inaba H, Yamada T, Hajika M, et al. Molecular basis of a shattering resistance boosting global dissemination of soybean. Proceedings of the National Academy of Sciences of the United States of America. 2014;**111**(50):17797-17802

[24] Dong Y, Yang X, Liu J, Wang B-H, Liu B-L, Wang Y-Z. Pod shattering resistance associated with domestication is mediated by a NAC gene in soybean. Nature Communications. 2014;**5**(1):3352

[25] Suanum W, Somta P, Kongjaimun A, Yimram T, Kaga A, Tomooka N, et al. Co-localization of QTLs for pod fiber content and pod shattering in F2 and backcross populations between yardlong bean and wild cowpea. Molecular Breeding. 2016;**36**(6):80

[26] Koinange EMK, Singh SP, Gepts P. Genetic control of the domestication syndrome in common bean. Crop Science. 1996;**36**:1037-1045

[27] Rau D, Murgia ML, Rodriguez M, Bitocchi E, Bellucci E, Fois D, et al. Genomic dissection of pod shattering in common bean: Mutations at non-orthologous loci at the basis of convergent phenotypic evolution under domestication of leguminous species. The Plant Journal. 2019;**97**(4):693-714

[28] Fourquin C, del Cerro C, Victoria FC, Vialette-Guiraud A, de Oliveira AC, Ferrándiz C. A change in SHATTERPROOF protein lies at the origin of a fruit morphological novelty and a new strategy for seed dispersal in Medicago genus. Plant Physiology. 2013;**162**(2):907-917

[29] Cormier F, Foulkes J, Hirel B, Gouache D, Moënne-Loccoz Y, Le Gouis J. Breeding for increased nitrogen-use efficiency: A review for wheat (T. aestivum L.). Plant Breeding. 2016;**135**(3):255-278

[30] Hu B, Wang W, Ou S, Tang J, Li H, Che R, et al. Variation in NRT1.1B contributes to nitrate-use divergence between rice subspecies. Nature Genetics. 2015 Jul;**47**(7):834-838

[31] Chen Z, Liu C, Wang Y, He T, Gao R, Xu H, et al. Expression analysis of nitrogen metabolism-related genes reveals differences in adaptation to lownitrogen stress between two different barley cultivars at seedling stage. International Journal of Genomics. 2018;**2018**:1-10

[32] Astier J, Gross I, Durner J. Nitric oxide production in plants: An update. Journal of Experimental Botany. 2018;**69**(14):3401-3411

[33] Lin CM, Koh S, Stacey G, Yu SM, Lin TY, Tsay YF. Cloning and functional characterization of a constitutively

**6**

*Recent Advances in Grain Crops Research*

[1] Belobrajdic DP, AR B. The potential role of phytochemicals in wholegrain cereals for the prevention of type-2 diabetes. Nutrition Journal.

[2] Wessel TJA, Values H. The

Agriculture Human Values.

Cereals. 2013;**4**(3):32-35

agricultural foundations of civilization.

[10] United Nations Department of Economics and Social Affairs. Population Division. Available from: https://www.un.org/en/development/ desa/population/index.asp [Accessed:

[11] Giraldo P, Benavente E,

Manzano-Agugliaro F, Gimenez EJA. Worldwide research trends on wheat and barley: A bibliometric comparative analysis. Agronomy. 2019;**9**(7):352

[12] International Barley Hub. Available from: http://www.barleyhub.org/ [Accessed: November 2019]

[13] Di Vittori V, Gioia T, Rodriguez M, Bellucci E, Bitocchi E, Nanni L, et al. Convergent evolution of the seed shattering trait. Genes (Basel).

[14] Liljegren SJ, Ditta GS, Eshed Y, Savidge B, Bowman JL, Yanofsky MF. SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis. Nature. 2000;**404**(6779):766-770

[15] Rajani S, Sundaresan V. The Arabidopsis myc/bHLH gene ALCATRAZ enables cell separation in fruit dehiscence. Current Biology.

[16] Mitsuda N, Ohme-Takagi M. NAC transcription factors NST1 and NST3 regulate pod shattering in a partially redundant manner by promoting secondary wall formation after the establishment of tissue identity. The Plant Journal. 2008;**56**(5):768-778

[17] Mitsuda N, Iwase A, Yamamoto H, Yoshida M, Seki M, Shinozaki K, et al. NAC transcription factors, NST1 and NST3, are key regulators of the formation of secondary walls in woody tissues of Arabidopsis. Plant Cell.

2001;**11**(24):1914-1922

2007;**19**(1):270-280

November 2019]

2019;**10**(1):1-16

[3] Hillman G, Hedges R, Moore A, Colledge S, Pettitt P. New evidence of Lateglacial cereal cultivation at Abu Hureyra on the Euphrates. The Holocene. 2001;**11**(4):383-393

[4] Sarwar MH, Sarwar MF, Sarwar M, Qadri NA, Moghal S. The importance of cereals (Poaceae: Gramineae) nutrition in human health: A review. Journal of

[5] Kumar V, Sinha AK, Makkar HP, De Boeck G, Becker K. Dietary roles of non-starch polysaccharides in human nutrition: A review. Critical Reviews in Food Science. 2012;**52**(10):899-935

[6] Jukanti AK, Gaur PM, Gowda C, Chibbar RN. Nutritional quality and health benefits of chickpea (*Cicer arietinum* L.): A review. British Journal of Nutrition. 2012;**108**(S1):S11-S26

[7] Iqbal A, Khalil IA, Ateeq N, MS K. Nutritional quality of important food legumes. Food Chemistry.

[8] Nkhata SG, Ayua E, Kamau EH, Shingiro JB. Fermentation and germination improve nutritional value of cereals and legumes through activation of endogenous enzymes. Food Science. 2018;**6**(8):2446-2458

[9] Wheat, Initiative. Available from: https://wwwwheatinitiativeorg/ [Accessed: November 2019]

2006;**97**(2):331-335

2013;**12**(1):62

**References**

1984;**1**(2):9-12

expressed nitrate transporter gene, OsNRT1, from rice. Plant Physiology. 2000;**122**(2):379-388

[34] Hu B, Wei W, Ou S, Tang J, Hua I, Che R, et al. Nature Genetics. 2015;**47**:834-838

[35] Fan X, Naz M, Fan X, Xuan W, Miller AJ, Xu G. Plant nitrate transporters: From gene function to application. Journal of Experimental Botany. 2017;**68**(10):2463-2475

[36] Leran S, Varala K, Boyer JC, Chiurazzi M, Crawford N, Daniel-Vedele F, et al. A unified nomenclature of NITRATE TRANSPORTER 1/ PEPTIDE TRANSPORTER family members in plants. Trends in Plant Science. 2014;**19**(1):5-9

[37] von Wittgenstein NJ, Le CH, Hawkins BJ, Ehlting J. Evolutionary classification of ammonium, nitrate, and peptide transporters in land plants. BMC Evolutionary Biology. 2014;**14**:11

[38] Komarova NY, Thor K, Gubler A, Meier S, Dietrich D, Weichert A, et al. AtPTR1 and AtPTR5 transport dipeptides in planta. Plant Physiology. 2008;**148**(2):856-869

[39] Krouk G, Lacombe B, Bielach A, Perrine-Walker F, Malinska K, Mounier E, et al. Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Developmental Cell. 2010;**18**(6):927-937

[40] Kanno Y, Hanada A, Chiba Y, Ichikawa T, Nakazawa M, Matsui M, et al. Identification of an abscisic acid transporter by functional screening using the receptor complex as a sensor. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(24):9653-9658

[41] Chiba Y, Shimizu T, Miyakawa S, Kanno Y, Koshiba T, Kamiya Y, et al.

Identification of Arabidopsis thaliana NRT1/PTR FAMILY (NPF) proteins capable of transporting plant hormones. Journal of Plant Research. 2015;**128**(4):679-686

[42] David LC, Berquin P, Kanno Y, Seo M, Daniel-Vedele F, Ferrario-Mery S. N availability modulates the role of NPF3.1, a gibberellin transporter, in GA-mediated phenotypes in Arabidopsis. Planta. 2016 Dec;**244**(6):1315-1328

[43] Zhao M, Geng X, Bi W, Xu Q, Sun J, Huang Y, et al. Recombination between DEP1 and NRT1.1B under japonica and indica genetic backgrounds to improve grain yield in rice. Euphytica. 2017;**213**(12):265

[44] Wang W, Hu B, Yuan D, Liu Y, Che R, Hu Y, et al. Expression of the nitrate transporter gene OsNRT1.1A/ OsNPF6.3 confers high yield and early maturation in Rice. The Plant Cell. 2018;**30**(3):638-651

[45] Zhang J, Liu YX, Zhang N, Hu B, Jin T, Xu H, et al. NRT1.1B is associated with root microbiota composition and nitrogen use in field-grown rice. Nature Biotechnology. 2019;**37**(6):676-684

[46] Hussain A, Imran QM, Yun B-W. CRISPR/Cas9-Mediated Gene Editing in Grain Crops. Recent Advances in Grain Crops Research: IntechOpen; 2019

**9**

**1. Introduction**

**Chapter 2**

**Abstract**

CRISPR/Cas9-Mediated Gene

*Adil Hussain, Qari Muhammad Imran and Byung-Wook Yun*

The development of reliable and efficient techniques for making precise targeted changes in the genome of living organisms has been a long-standing objective of researchers throughout the world. In plants, different methods, each with several different variations, have been developed for this purpose, though many of them are hampered either by providing only temporary modification of gene function or unpredictable off-target results. The recent discovery of clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated 9 (Cas9) nucleases started a new era in genome editing. Basically, the CRISPR/Cas system is a natural immune response of prokaryotes to resist foreign genetic elements entering via plasmids and phages. Through this naturally occurring gene editing system, bacteria create DNA segments known as CRISPR arrays that allow them to "remember" foreign genetic material for protection against it and other similar sequences in the future. This system has now been adopted by researchers in laboratory to create a short guide RNA that binds to specific target sequences of DNA in eukaryotic genome, and the Cas9 enzyme cuts the DNA at the targeted location. Once cut, the cell's endogenous DNA repair machinery is used to add, delete, or replace pieces of genetic material. Though CRISPR/Cas9 technology has been recently developed, it has started to be regularly used for gene editing in plants as well as animals to good success. It has been proved as an efficient transgene-free technique. A simple search on PubMed (NCBI) shows that among all plants, 80 different studies published since 2013 involved CRISPR/Cas9-mediated genome editing in rice. Of these, 20, 13, and 24 papers have been published in 2019, 2018, and 2017, respectively. Furthermore, 20 different studies published since 2014 utilized CRISPR/Cas9 system for gene editing in wheat, where five of these studies were published in 2019 and seven were published in 2018. Genomes of other grain crops edited through this technique include maize, sorghum, barley, etc. This indicates the high utility of this technique for gene editing in grain crops. Here we emphasize on CRISPR/Cas9-

Editing in Grain Crops

mediated gene editing in rice, wheat, and maize.

**Keywords:** gene editing, cereal crops, CRISPR/Cas9, rice, wheat, maize

Perhaps one of the most important differences that can define humans apart from others in the animal kingdom is the ability to make choices among good, better, and best. The ability to reason, argue, research, and adopt what is (or at least what *he* thinks is) best for him. Plants, being the primary producers on this planet, have always been the most important resource of life for the human omnivores. We

#### **Chapter 2**

*Recent Advances in Grain Crops Research*

expressed nitrate transporter gene, OsNRT1, from rice. Plant Physiology. Identification of Arabidopsis thaliana NRT1/PTR FAMILY (NPF) proteins capable of transporting plant

hormones. Journal of Plant Research.

[42] David LC, Berquin P, Kanno Y, Seo M, Daniel-Vedele F, Ferrario-Mery S. N availability modulates the role of NPF3.1, a gibberellin transporter, in GA-mediated phenotypes in Arabidopsis. Planta. 2016

[43] Zhao M, Geng X, Bi W, Xu Q, Sun J, Huang Y, et al. Recombination between DEP1 and NRT1.1B under japonica and indica genetic backgrounds to improve grain yield in rice. Euphytica.

[44] Wang W, Hu B, Yuan D, Liu Y, Che R, Hu Y, et al. Expression of the nitrate transporter gene OsNRT1.1A/ OsNPF6.3 confers high yield and early maturation in Rice. The Plant Cell.

[45] Zhang J, Liu YX, Zhang N, Hu B, Jin T, Xu H, et al. NRT1.1B is associated with root microbiota composition and nitrogen use in field-grown rice. Nature Biotechnology. 2019;**37**(6):676-684

[46] Hussain A, Imran QM, Yun B-W. CRISPR/Cas9-Mediated Gene Editing in Grain Crops. Recent Advances in Grain Crops Research: IntechOpen; 2019

2015;**128**(4):679-686

Dec;**244**(6):1315-1328

2017;**213**(12):265

2018;**30**(3):638-651

[34] Hu B, Wei W, Ou S, Tang J, Hua I, Che R, et al. Nature Genetics.

[35] Fan X, Naz M, Fan X, Xuan W, Miller AJ, Xu G. Plant nitrate transporters: From gene function to application. Journal of Experimental Botany. 2017;**68**(10):2463-2475

[36] Leran S, Varala K, Boyer JC, Chiurazzi M, Crawford N, Daniel-Vedele F, et al. A unified nomenclature of NITRATE TRANSPORTER 1/ PEPTIDE TRANSPORTER family members in plants. Trends in Plant

[37] von Wittgenstein NJ, Le CH, Hawkins BJ, Ehlting J. Evolutionary classification of ammonium, nitrate, and peptide transporters in land plants. BMC Evolutionary Biology. 2014;**14**:11

[38] Komarova NY, Thor K, Gubler A, Meier S, Dietrich D, Weichert A, et al. AtPTR1 and AtPTR5 transport

dipeptides in planta. Plant Physiology.

[39] Krouk G, Lacombe B, Bielach A, Perrine-Walker F, Malinska K, Mounier E, et al. Nitrate-regulated auxin transport by NRT1.1 defines a mechanism for nutrient sensing in plants. Developmental Cell.

[40] Kanno Y, Hanada A, Chiba Y, Ichikawa T, Nakazawa M, Matsui M, et al. Identification of an abscisic acid transporter by functional screening using the receptor complex as a sensor. Proceedings of the National Academy of Sciences of the United States of America. 2012;**109**(24):9653-9658

[41] Chiba Y, Shimizu T, Miyakawa S, Kanno Y, Koshiba T, Kamiya Y, et al.

Science. 2014;**19**(1):5-9

2008;**148**(2):856-869

2010;**18**(6):927-937

2000;**122**(2):379-388

2015;**47**:834-838

**8**

## CRISPR/Cas9-Mediated Gene Editing in Grain Crops

*Adil Hussain, Qari Muhammad Imran and Byung-Wook Yun*

#### **Abstract**

The development of reliable and efficient techniques for making precise targeted changes in the genome of living organisms has been a long-standing objective of researchers throughout the world. In plants, different methods, each with several different variations, have been developed for this purpose, though many of them are hampered either by providing only temporary modification of gene function or unpredictable off-target results. The recent discovery of clustered regularly interspaced short palindromic repeats (CRISPRs) and the CRISPR-associated 9 (Cas9) nucleases started a new era in genome editing. Basically, the CRISPR/Cas system is a natural immune response of prokaryotes to resist foreign genetic elements entering via plasmids and phages. Through this naturally occurring gene editing system, bacteria create DNA segments known as CRISPR arrays that allow them to "remember" foreign genetic material for protection against it and other similar sequences in the future. This system has now been adopted by researchers in laboratory to create a short guide RNA that binds to specific target sequences of DNA in eukaryotic genome, and the Cas9 enzyme cuts the DNA at the targeted location. Once cut, the cell's endogenous DNA repair machinery is used to add, delete, or replace pieces of genetic material. Though CRISPR/Cas9 technology has been recently developed, it has started to be regularly used for gene editing in plants as well as animals to good success. It has been proved as an efficient transgene-free technique. A simple search on PubMed (NCBI) shows that among all plants, 80 different studies published since 2013 involved CRISPR/Cas9-mediated genome editing in rice. Of these, 20, 13, and 24 papers have been published in 2019, 2018, and 2017, respectively. Furthermore, 20 different studies published since 2014 utilized CRISPR/Cas9 system for gene editing in wheat, where five of these studies were published in 2019 and seven were published in 2018. Genomes of other grain crops edited through this technique include maize, sorghum, barley, etc. This indicates the high utility of this technique for gene editing in grain crops. Here we emphasize on CRISPR/Cas9 mediated gene editing in rice, wheat, and maize.

**Keywords:** gene editing, cereal crops, CRISPR/Cas9, rice, wheat, maize

#### **1. Introduction**

Perhaps one of the most important differences that can define humans apart from others in the animal kingdom is the ability to make choices among good, better, and best. The ability to reason, argue, research, and adopt what is (or at least what *he* thinks is) best for him. Plants, being the primary producers on this planet, have always been the most important resource of life for the human omnivores. We

have always chosen, selected, and adopted plant species that produced either more or good; and hence, driving *plant domestication* to this day. Initially, this domestication was only based upon observations, personal, physical likes and dislikes. Later, it was based on more complex characteristics that tempted the crossing of different plants with desirable characters to make a new variety or cultivar carrying multiple desirable characters. Such classical or conventional breeding methods included selection and hybridization. Afterward, using DNA-level information, scientists used these methods to tag a large number of quantitative trait loci (QTLs) via marker-assisted breeding or selection for combining different traits in a single variety.

With further developments in genetic engineering, genome modification studies were performed in plants [1]. This was specifically aided by the discovery of nuclease enzyme, the utilization of different bacterial plasmids for cloning of target genes in bacteria as well as target plants and by the development of plasmid vectors carrying antibiotic resistance genes, a variety of constitutive, inducible as well as tissue-specific promoters, and different reporter genes that made the cloning and transformation easier. The discovery of the Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently that of the clustered regularly interspaced short palindromic repeats (CRISPRs) in genomes has further aided the genome editing techniques in different plant species.

#### **1.1 Genome editing in nature**

Genome editing involves molecular tools that lead to targeted modification of specific DNA sequences inside the genome. These are usually based on the production of double strand brakes (DSBs) at the specific DNA sites that trigger DNA repair of the cell. In plant cells, this generally happens through the process of nonhomologous end-joining commonly abbreviated as NHEJ. However, this process is error-prone, thus exploited by scientists to target genes for possible modification. Sometimes, plants repair the DSBs through homologous recombination which is error-free. Hence, it could be used by scientists to precisely edit DNA or insert DNA sequences in a given genome.

Genetic modification or genome editing is already in practice in cells of living organisms and is one of the most magnificent specters of nature. Natural DNA repair mechanisms, losing and acquiring of genetic material in bacteria, transmission of bacteriophages through bacterial transduction, and genetic modification of plant genome by Ti plasmid during infection by *Agrobacterium tumefaciens* are some of the examples of genetic modifications from nature. DSBs have also been shown to be induced naturally by different factors such as various reactive oxygen species (ROS), radiation, and others. Intricate and complex interconnected cellular networks drive post-transcriptional RNA modifications and post-translational protein modifications to accommodate a literally unlimited number of functions from a limited set of genes.

In the last few decades by unraveling the details of mechanisms underlying natural genome editing, scientists have shown that such editing can also be induced by synthetic nucleases and more recently by the CRIPR system adopted from bacterial defense system which is one of the most promising gene editing technologies introduced in 2012.

#### **1.2 The CRISPR/Cas system**

Among the different types of defense systems found in prokaryotes and eukaryotes, a unique molecular system known as the CRISPR/Cas not only provides

**11**

*CRISPR/Cas9-Mediated Gene Editing in Grain Crops DOI: http://dx.doi.org/10.5772/intechopen.88115*

subsequent implications of such studies.

guide RNA (sgRNA) [5].

protection against genetic attack, but also keeps a record or genetic memory of such attacks for future safety. This technique has been employed in several different studies by scientists from around the world for targeted genetic modifications in a plethora of living organisms, but perhaps most ambitiously by the Chinese scientists who not only used the technique for gene editing an armada of plant and animal species but also humans (https://www.nature.com/articles/d41586-019- 00673-1), which raises certain ethical concerns about the practical application and

A key factor for sustainable agriculture is improvement of crops via genetic engineering. It is true that DNA-free editing techniques are now desirable for molecular breeding in crops for which CRISPR/Cas may offer a better option for more complex and controlled genetic rearrangements. CRISPR arrays are characterized by a series of 20- to 50-bp genomic loci, which are unique spacers separated by direct repeats that usually have similar length to preceding AT-rich fragments [2]. CRISPR loci were discovered about two decades ago in *Escherichia coli* following viral infection as part of the immune system through which the bacteria keep a genetic memory of the viral genome fragments in order to defend against future infections if any other foreign DNA sequences match the unique spacer sequences in the memory (also known as protospacers) [2, 3]. Briefly speaking about the chief attributes of the mechanism of action, CRISPR-based defense system can be divided into three distinct events: adaptation, biogenesis of CRISPR-RNAs, and interference. Upon the introduction of a foreign DNA, the machinery selects protospacers that are inserted into the CRISPR locus following which CRISPR-RNAs (CrRNAs) are synthesized through a series of events, each with a corresponding spacer. Then, the Cas proteins interact with CrRNAs to initiate interference and consequent degradation of the foreign DNA by endonucleases [4]. It is now clear that CRISPR/Cas9 action is based on the participation of Cas9 protein and single

Though CRISPR/Cas9 technology is relatively recent, it has started to be regularly used for gene editing in plants as well as animals to good success. It has been proved to be an efficient transgene-free technique. A simple search on PubMed (NCBI) shows that among all plants, 80 different studies published since 2013 involved CRISPR/Cas9-mediated genome editing in rice. Of these, 20, 13, and 24 papers have been published in 2019, 2018, and 2017, respectively. Furthermore, 20 different studies published since 2014 utilized CRISPR/Cas9 system for gene editing in wheat, where five of these studies were published in 2019 and seven were published in 2018. Genomes of other grain crops edited through this technique include maize, sorghum, barley etc. This indicates the high utility of this technique for gene editing in grain crops. Evidence that CRISPR/Cas9 system can be used for gene editing came around 2013 [6, 7] and the next paper showing the use of this technique for gene editing in rice and wheat was published in 2014 [8]. Furthermore, other studies published from 2013 to 2015 showed the use of this technique for gene editing in an array of organisms such as yeast, zebrafish, fruit flies, mosquitoes, nematodes, mice, monkeys, and human embryos, and several plant species, indicat-

ing sufficiently fast and easy application of this technique in this crop.

**1.3 CRISPR/Cas9 for crop improvement in rice, wheat, and maize**

The importance of agriculture for human survival cannot be questioned. As mentioned earlier, plants are the only primary producers on planet earth providing food, fiber, and other raw material generating the bulk of energy required for the growth of human population. However, plants/crops are facing several challenges and now their own survival may well be at stake. In this context, classical breeding

#### *CRISPR/Cas9-Mediated Gene Editing in Grain Crops DOI: http://dx.doi.org/10.5772/intechopen.88115*

*Recent Advances in Grain Crops Research*

**1.1 Genome editing in nature**

sequences in a given genome.

limited set of genes.

introduced in 2012.

**1.2 The CRISPR/Cas system**

variety.

have always chosen, selected, and adopted plant species that produced either more or good; and hence, driving *plant domestication* to this day. Initially, this domestication was only based upon observations, personal, physical likes and dislikes. Later, it was based on more complex characteristics that tempted the crossing of different plants with desirable characters to make a new variety or cultivar carrying multiple desirable characters. Such classical or conventional breeding methods included selection and hybridization. Afterward, using DNA-level information, scientists used these methods to tag a large number of quantitative trait loci (QTLs) via marker-assisted breeding or selection for combining different traits in a single

With further developments in genetic engineering, genome modification studies were performed in plants [1]. This was specifically aided by the discovery of nuclease enzyme, the utilization of different bacterial plasmids for cloning of target genes in bacteria as well as target plants and by the development of plasmid vectors carrying antibiotic resistance genes, a variety of constitutive, inducible as well as tissue-specific promoters, and different reporter genes that made the cloning and transformation easier. The discovery of the Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently that of the clustered regularly interspaced short palindromic repeats (CRISPRs) in genomes has further aided the genome editing techniques in different plant species.

Genome editing involves molecular tools that lead to targeted modification of specific DNA sequences inside the genome. These are usually based on the production of double strand brakes (DSBs) at the specific DNA sites that trigger DNA repair of the cell. In plant cells, this generally happens through the process of nonhomologous end-joining commonly abbreviated as NHEJ. However, this process is error-prone, thus exploited by scientists to target genes for possible modification. Sometimes, plants repair the DSBs through homologous recombination which is error-free. Hence, it could be used by scientists to precisely edit DNA or insert DNA

Genetic modification or genome editing is already in practice in cells of living organisms and is one of the most magnificent specters of nature. Natural DNA repair mechanisms, losing and acquiring of genetic material in bacteria, transmission of bacteriophages through bacterial transduction, and genetic modification of plant genome by Ti plasmid during infection by *Agrobacterium tumefaciens* are some of the examples of genetic modifications from nature. DSBs have also been shown to be induced naturally by different factors such as various reactive oxygen species (ROS), radiation, and others. Intricate and complex interconnected cellular networks drive post-transcriptional RNA modifications and post-translational protein modifications to accommodate a literally unlimited number of functions from a

In the last few decades by unraveling the details of mechanisms underlying natural genome editing, scientists have shown that such editing can also be induced by synthetic nucleases and more recently by the CRIPR system adopted from bacterial defense system which is one of the most promising gene editing technologies

Among the different types of defense systems found in prokaryotes and eukaryotes, a unique molecular system known as the CRISPR/Cas not only provides

**10**

protection against genetic attack, but also keeps a record or genetic memory of such attacks for future safety. This technique has been employed in several different studies by scientists from around the world for targeted genetic modifications in a plethora of living organisms, but perhaps most ambitiously by the Chinese scientists who not only used the technique for gene editing an armada of plant and animal species but also humans (https://www.nature.com/articles/d41586-019- 00673-1), which raises certain ethical concerns about the practical application and subsequent implications of such studies.

A key factor for sustainable agriculture is improvement of crops via genetic engineering. It is true that DNA-free editing techniques are now desirable for molecular breeding in crops for which CRISPR/Cas may offer a better option for more complex and controlled genetic rearrangements. CRISPR arrays are characterized by a series of 20- to 50-bp genomic loci, which are unique spacers separated by direct repeats that usually have similar length to preceding AT-rich fragments [2]. CRISPR loci were discovered about two decades ago in *Escherichia coli* following viral infection as part of the immune system through which the bacteria keep a genetic memory of the viral genome fragments in order to defend against future infections if any other foreign DNA sequences match the unique spacer sequences in the memory (also known as protospacers) [2, 3]. Briefly speaking about the chief attributes of the mechanism of action, CRISPR-based defense system can be divided into three distinct events: adaptation, biogenesis of CRISPR-RNAs, and interference. Upon the introduction of a foreign DNA, the machinery selects protospacers that are inserted into the CRISPR locus following which CRISPR-RNAs (CrRNAs) are synthesized through a series of events, each with a corresponding spacer. Then, the Cas proteins interact with CrRNAs to initiate interference and consequent degradation of the foreign DNA by endonucleases [4]. It is now clear that CRISPR/Cas9 action is based on the participation of Cas9 protein and single guide RNA (sgRNA) [5].

Though CRISPR/Cas9 technology is relatively recent, it has started to be regularly used for gene editing in plants as well as animals to good success. It has been proved to be an efficient transgene-free technique. A simple search on PubMed (NCBI) shows that among all plants, 80 different studies published since 2013 involved CRISPR/Cas9-mediated genome editing in rice. Of these, 20, 13, and 24 papers have been published in 2019, 2018, and 2017, respectively. Furthermore, 20 different studies published since 2014 utilized CRISPR/Cas9 system for gene editing in wheat, where five of these studies were published in 2019 and seven were published in 2018. Genomes of other grain crops edited through this technique include maize, sorghum, barley etc. This indicates the high utility of this technique for gene editing in grain crops. Evidence that CRISPR/Cas9 system can be used for gene editing came around 2013 [6, 7] and the next paper showing the use of this technique for gene editing in rice and wheat was published in 2014 [8]. Furthermore, other studies published from 2013 to 2015 showed the use of this technique for gene editing in an array of organisms such as yeast, zebrafish, fruit flies, mosquitoes, nematodes, mice, monkeys, and human embryos, and several plant species, indicating sufficiently fast and easy application of this technique in this crop.

#### **1.3 CRISPR/Cas9 for crop improvement in rice, wheat, and maize**

The importance of agriculture for human survival cannot be questioned. As mentioned earlier, plants are the only primary producers on planet earth providing food, fiber, and other raw material generating the bulk of energy required for the growth of human population. However, plants/crops are facing several challenges and now their own survival may well be at stake. In this context, classical breeding

methods may not be suitable for increasing per unit area production relatively quickly. Hence, the rational use of biotechnological tools is of paramount importance. Editing crop genomes is a promising technique to cope with agricultural challenges. Development of these methodologies is useful for genetic improvement of crops [9]. In 2013, the pioneering works published by Lit et al. using *Arabidopsis* [10], Nekrasov et al. using tobacco [11], and Shan et al. using rice and wheat [12] demonstrated the suitability of the CRISPR/Cas9 system for crop genetic improvement. Since then, a plethora of research on crop genome editing via CRISPR/ Cas9 has been published. Crops targeted for genetic editing via this system include soybean [13–15], tomato [16–19], potato [20], cucumber [21], maize [22, 23], the oil-seed plant camellia sativa [24], grapes and apple [25], *Brassica oleracea* and barley [26], watermelon [27], sweet orange [28], *Populus tomentosa* [29], and others to various ends.

The maize ARGOS8 variants generated via CRISPR/Cas9 showed significantly improved yield under drought stress conditions [30].

Rice, given its worldwide relevance for food, has been one of the most studied crops in terms of CRISPR/Cas9 application [31–36]. These include genetic modification for increased disease resistance [37], and herbicide resistance [38]. At present, more than 80 different research papers have been published using CRISPRbased genetic editing in rice and more than 15 papers involving wheat. These studies involve applicative use of CRISPR in rice. Li et al. [39] developed photo- and thermo-sensitive male sterile rice lines using this technique to exploit heterosis and speed up breeding [40]. Rice cultivars carrying high genetic resistance to the rice blast disease have been recently developed by Wang et al. [37]. Li et al. [41] at the South China Normal University, China, targeted four yield-related genes *Gn1a*, *DEP1*, *GS3*, and *IPA1* in the rice genome for modification via CRISPR/Cas9 for the improvement of different agronomic traits such as grain number and size, panicle structure indicating the application of this technique for improvement of agronomic traits in rice. Furthermore, the much required herbicide resistance in rice has also been genetically enhanced through CRISPR/Cas9-mediated gene editing [38]. More interestingly, the use of Cpf1 which is an endonuclease (alternative to Cas9) has been successfully used for targeted gene editing in rice and as a transcriptional regulator [42, 43]. More recently, a research group from University of Arkansas, USA*,* developed a soybean heat shock-inducible promoter CRISPR/Cas9 system for heritable mutations in rice. They transformed the HS-CRISPR/Cas9 vectors in rice and found only a low rate of target mutations before induction by heat shock compared to an increased rate of target mutations after heat shock treatment indicating that CRISPR/Cas9 is a controlled and efficient platform for gene editing in rice. This work is available online as *Pre-print* at BioRxiv [44]. Xu et al. in 2016 [45] mutated three rice genes *GW2*, *GW5*, and *TGW6* to increase seed size by up to 30% in the triple mutant lines.

Both durum and bread wheat have also been the subject of successful CRISPR/ Cas9-mediated genetic modification for powdery mildew resistance and other objectives [42, 46–48]. However, in wheat, regeneration of plants from CRISPRedited protoplasts has been difficult [12]. In addition, the complexity of wheat genome together with time-consuming tissue culture techniques has made it difficult for scientists to undergo ambitious genome editing projects via CRISPR/Cas9 in this important cereal. Researchers at the Chinese Academy of Science obtained the first CRISPR/Cas9-edited wheat plants [48] by editing three homoeoalleles of a hexaploid bread wheat cultivar to confer heritable resistance against powdery mildew of wheat. Later, the same research group obtained transgene-free genomeedited wheat plants using transient expression of CRISPR/Cas9 DNA or RNA [46]. In 2018, Wang et al. [49] knocked out all the wheat homologs of the rice *TaGW2*

**13**

**Table 1.**

*List of studies involving CRISPR/Cas9-mediated gene editing in grain crops.*

*CRISPR/Cas9-Mediated Gene Editing in Grain Crops DOI: http://dx.doi.org/10.5772/intechopen.88115*

(a gene that negatively regulates seed size) in order to significantly increase wheat kernel size. Transgene-free low-gluten wheat has been developed by Sánchez-León et al. via CRISPR/Cas9-mediated genetic engineering [50]. This indicates that CRISPR/Cas9 can be successfully applied to engineer cereal crops for higher yields. In their book chapter, Chilcoat et al. [51] discuss the use of CRISPR/Cas9 for crop improvement in maize and soybean and have discussed the molecular details of gene editing projects via CRISPR/Cas9 such as those involving *WX1*, *ALS*, and *ARGOS8* genes in maize. Different studies report successful modification of maize genome using CRISPR/Cas technology [22, 23, 30, 52] for editing traits such as

**S. no. Plant/fungus Gene/locus Citation** Rice blast fungus (*Pyricularia oryzae)* RNAP II [57] Rice (*Oryza sativa*) *TMS5* [58] MPK1 and MPK6 [59] HAK1 [60] *CDKA1*, *CDKA2* and *CDKB1* [32] *IAA23* [61] EPSPS [62] *GN1A*, *DEP1*, *GS3* and *IPA1* [41] *GBSSI* [63] *OsAPL2* and *OsAPS2b* [64] *TOS17* [65] *SBEI* and *SBEIIb* [66] *CALD5H1* [67] *NRAMP5* [68] *ERF922* [37] *EPFL9* [69] Wheat (*Triticum aestivum*) *DREB2* and *ERF3* [70] *LOX2* and *UBIL1* [71] 19 *MS1* [72] α-*GLIADINS* [50] *MS45* [73] Maize (*Zea mays*) *AGO18A* and *AGO18B* [22] 23 *MS8* [49] *DMC1* [74] 25 *ZB7* [75] *ARGOS8* [76] Barley (*Hordeum vulgare*) *CKX1*, *CKX3* [77] *PAPHY* [78] *ENGASE* [79] Sorghum (*Sorghum bicolor*) *CAD* and *PDS* [80]

#### *CRISPR/Cas9-Mediated Gene Editing in Grain Crops DOI: http://dx.doi.org/10.5772/intechopen.88115*

*Recent Advances in Grain Crops Research*

to various ends.

methods may not be suitable for increasing per unit area production relatively quickly. Hence, the rational use of biotechnological tools is of paramount importance. Editing crop genomes is a promising technique to cope with agricultural challenges. Development of these methodologies is useful for genetic improvement of crops [9]. In 2013, the pioneering works published by Lit et al. using *Arabidopsis* [10], Nekrasov et al. using tobacco [11], and Shan et al. using rice and wheat [12] demonstrated the suitability of the CRISPR/Cas9 system for crop genetic improvement. Since then, a plethora of research on crop genome editing via CRISPR/ Cas9 has been published. Crops targeted for genetic editing via this system include soybean [13–15], tomato [16–19], potato [20], cucumber [21], maize [22, 23], the oil-seed plant camellia sativa [24], grapes and apple [25], *Brassica oleracea* and barley [26], watermelon [27], sweet orange [28], *Populus tomentosa* [29], and others

The maize ARGOS8 variants generated via CRISPR/Cas9 showed significantly

Rice, given its worldwide relevance for food, has been one of the most studied crops in terms of CRISPR/Cas9 application [31–36]. These include genetic modification for increased disease resistance [37], and herbicide resistance [38]. At present, more than 80 different research papers have been published using CRISPRbased genetic editing in rice and more than 15 papers involving wheat. These studies involve applicative use of CRISPR in rice. Li et al. [39] developed photo- and thermo-sensitive male sterile rice lines using this technique to exploit heterosis and speed up breeding [40]. Rice cultivars carrying high genetic resistance to the rice blast disease have been recently developed by Wang et al. [37]. Li et al. [41] at the South China Normal University, China, targeted four yield-related genes *Gn1a*, *DEP1*, *GS3*, and *IPA1* in the rice genome for modification via CRISPR/Cas9 for the improvement of different agronomic traits such as grain number and size, panicle structure indicating the application of this technique for improvement of agronomic traits in rice. Furthermore, the much required herbicide resistance in rice has also been genetically enhanced through CRISPR/Cas9-mediated gene editing [38]. More interestingly, the use of Cpf1 which is an endonuclease (alternative to Cas9) has been successfully used for targeted gene editing in rice and as a transcriptional regulator [42, 43]. More recently, a research group from University of Arkansas, USA*,* developed a soybean heat shock-inducible promoter CRISPR/Cas9 system for heritable mutations in rice. They transformed the HS-CRISPR/Cas9 vectors in rice and found only a low rate of target mutations before induction by heat shock compared to an increased rate of target mutations after heat shock treatment indicating that CRISPR/Cas9 is a controlled and efficient platform for gene editing in rice. This work is available online as *Pre-print* at BioRxiv [44]. Xu et al. in 2016 [45] mutated three rice genes *GW2*, *GW5*, and *TGW6* to increase seed size by up to 30% in the

Both durum and bread wheat have also been the subject of successful CRISPR/ Cas9-mediated genetic modification for powdery mildew resistance and other objectives [42, 46–48]. However, in wheat, regeneration of plants from CRISPRedited protoplasts has been difficult [12]. In addition, the complexity of wheat genome together with time-consuming tissue culture techniques has made it difficult for scientists to undergo ambitious genome editing projects via CRISPR/Cas9 in this important cereal. Researchers at the Chinese Academy of Science obtained the first CRISPR/Cas9-edited wheat plants [48] by editing three homoeoalleles of a hexaploid bread wheat cultivar to confer heritable resistance against powdery mildew of wheat. Later, the same research group obtained transgene-free genomeedited wheat plants using transient expression of CRISPR/Cas9 DNA or RNA [46]. In 2018, Wang et al. [49] knocked out all the wheat homologs of the rice *TaGW2*

improved yield under drought stress conditions [30].

**12**

triple mutant lines.

(a gene that negatively regulates seed size) in order to significantly increase wheat kernel size. Transgene-free low-gluten wheat has been developed by Sánchez-León et al. via CRISPR/Cas9-mediated genetic engineering [50]. This indicates that CRISPR/Cas9 can be successfully applied to engineer cereal crops for higher yields.

In their book chapter, Chilcoat et al. [51] discuss the use of CRISPR/Cas9 for crop improvement in maize and soybean and have discussed the molecular details of gene editing projects via CRISPR/Cas9 such as those involving *WX1*, *ALS*, and *ARGOS8* genes in maize. Different studies report successful modification of maize genome using CRISPR/Cas technology [22, 23, 30, 52] for editing traits such as

#### **Table 1.**

*List of studies involving CRISPR/Cas9-mediated gene editing in grain crops.*

lignin biosynthesis, sterility, and herbicide resistance, secondary metabolism, RNA metabolism, drought tolerance, and agronomic traits [51]. Male sterility in maize provides a useful tool to harness hybrid vigor. Therefore, generation of male sterile mutant lines is considered to be of paramount importance in the hybrid seed production industry. Chen et al. in 2018 [49] used CRISPR/Cas9 to target the *MS8* gene of maize and obtained transgene-free *ms8* male sterile plants in the F2 generation that could be used for crossing with other elite lines for hybrid seed production. Zhu et al. in 1999 [53] manipulated maize genes AHAs108 and AHA109 encoding acetohydroxyacid synthase enzyme using chimeric RNA/DNA oligonucleotides. Similar studies were performed for targeting various genes in cereal crops such as maize, rice, wheat, and barley [54–56].

**Table 1** summarizes the list of important studies involving CRISPR/Cas9 mediated gene editing in grain crops.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Adil Hussain1 \*, Qari Muhammad Imran<sup>2</sup> and Byung-Wook Yun<sup>2</sup> \*

1 Department of Agriculture, Abdul Wali Khan University, Mardan, Khyber Pakhtunkhwa, Pakistan

2 School of Applied Biosciences, College of Agriculture and Life Sciences, Kyungpook National University, Republic of Korea

\*Address all correspondence to<EMAIL_ADDRESS>and<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**15**

*CRISPR/Cas9-Mediated Gene Editing in Grain Crops DOI: http://dx.doi.org/10.5772/intechopen.88115*

[1] Puchta H, Fauser F. Gene targeting in plants: 25 years later. The International Journal of Developmental Biology.

Bush J, et al. Multiplex and homologous recombination–mediated genome editing in *Arabidopsis* and *Nicotiana benthamiana* using guide RNA and Cas9. Nature Biotechnology. 2013;**31**(8):688

Weigel D, Jones JD, Kamoun S. Targeted

[11] Nekrasov V, Staskawicz B,

mutagenesis in the model plant *Nicotiana benthamiana* using Cas9 RNA-guided endonuclease. Nature Biotechnology. 2013;**31**(8):691

[12] Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nature Biotechnology. 2013;**31**(8):686

[13] Sun X, Hu Z, Chen R, Jiang Q, Song G, Zhang H, et al. Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Scientific

Reports. 2015;**5**:10342

2014;**166**(3):1292-1297

2016;**6**:24765

2015;**15**(1):16

[14] Jacobs TB, LaFayette PR, Schmitz RJ, Parrott WA. Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnology.

[15] Cai Y, Chen L, Liu X, Sun S, Wu C, Jiang B, et al. CRISPR/Cas9-mediated genome editing in soybean hairy roots. PLoS One. 2015;**10**(8):e0136064

[16] Brooks C, Nekrasov V, Lippman ZB, Van Eck J. Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPRassociated9 system. Plant Physiology.

[17] Pan C, Ye L, Qin L, Liu X, He Y, Wang J, et al. CRISPR/Cas9-mediated efficient and heritable targeted

mutagenesis in tomato plants in the first and later generations. Scientific Reports.

[2] Kunin V, Sorek R, Hugenholtz P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biology. 2007;**8**(4):R61

[3] Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V. Editing plant genomes with CRISPR/ Cas9. Current Opinion in Biotechnology.

[4] Wright AV, Nuñez JK, Doudna JA. Biology and applications of CRISPR systems: Harnessing nature's toolbox for genome engineering. Cell.

[5] Liu X, Wu S, Xu J, Sui C, Wei J. Application of CRISPR/Cas9 in plant biology. Acta Pharmaceutica Sinica B.

[6] Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided

human genome engineering via Cas9. Science (New York, N.Y.).

[7] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex

[8] Shan Q, Wang Y, Li J, Gao C. Genome editing in rice and wheat using the CRISPR/Cas system. Nature Protocols. 2014;**9**(10):2395-2410

[9] Huang S, Weigel D, Beachy RN, Li J. A proposed regulatory framework for genome-edited crops. Nature

Genetics. 2016;**48**(2):109

[10] Li J-F, Norville JE, Aach J, McCormack M, Zhang D,

2013;**339**(6121):823-826

genome engineering using CRISPR/Cas systems. Science. 2013;**339**(6121):819-823

**References**

2015;**32**:76-84

2016;**164**(1-2):29-44

2017;**7**(3):292-302

2013;**57**(6-8):629-637

*CRISPR/Cas9-Mediated Gene Editing in Grain Crops DOI: http://dx.doi.org/10.5772/intechopen.88115*

#### **References**

*Recent Advances in Grain Crops Research*

maize, rice, wheat, and barley [54–56].

mediated gene editing in grain crops.

The authors declare no conflict of interest.

**Conflict of interest**

**14**

**Author details**

Khyber Pakhtunkhwa, Pakistan

\*, Qari Muhammad Imran<sup>2</sup>

Kyungpook National University, Republic of Korea

provided the original work is properly cited.

1 Department of Agriculture, Abdul Wali Khan University, Mardan,

2 School of Applied Biosciences, College of Agriculture and Life Sciences,

\*Address all correspondence to<EMAIL_ADDRESS>and<EMAIL_ADDRESS>
© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

lignin biosynthesis, sterility, and herbicide resistance, secondary metabolism, RNA metabolism, drought tolerance, and agronomic traits [51]. Male sterility in maize provides a useful tool to harness hybrid vigor. Therefore, generation of male sterile mutant lines is considered to be of paramount importance in the hybrid seed production industry. Chen et al. in 2018 [49] used CRISPR/Cas9 to target the *MS8* gene of maize and obtained transgene-free *ms8* male sterile plants in the F2 generation that could be used for crossing with other elite lines for hybrid seed production. Zhu et al. in 1999 [53] manipulated maize genes AHAs108 and AHA109 encoding acetohydroxyacid synthase enzyme using chimeric RNA/DNA oligonucleotides. Similar studies were performed for targeting various genes in cereal crops such as

**Table 1** summarizes the list of important studies involving CRISPR/Cas9-

and Byung-Wook Yun<sup>2</sup>

\*

Adil Hussain1

[1] Puchta H, Fauser F. Gene targeting in plants: 25 years later. The International Journal of Developmental Biology. 2013;**57**(6-8):629-637

[2] Kunin V, Sorek R, Hugenholtz P. Evolutionary conservation of sequence and secondary structures in CRISPR repeats. Genome Biology. 2007;**8**(4):R61

[3] Belhaj K, Chaparro-Garcia A, Kamoun S, Patron NJ, Nekrasov V. Editing plant genomes with CRISPR/ Cas9. Current Opinion in Biotechnology. 2015;**32**:76-84

[4] Wright AV, Nuñez JK, Doudna JA. Biology and applications of CRISPR systems: Harnessing nature's toolbox for genome engineering. Cell. 2016;**164**(1-2):29-44

[5] Liu X, Wu S, Xu J, Sui C, Wei J. Application of CRISPR/Cas9 in plant biology. Acta Pharmaceutica Sinica B. 2017;**7**(3):292-302

[6] Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science (New York, N.Y.). 2013;**339**(6121):823-826

[7] Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;**339**(6121):819-823

[8] Shan Q, Wang Y, Li J, Gao C. Genome editing in rice and wheat using the CRISPR/Cas system. Nature Protocols. 2014;**9**(10):2395-2410

[9] Huang S, Weigel D, Beachy RN, Li J. A proposed regulatory framework for genome-edited crops. Nature Genetics. 2016;**48**(2):109

[10] Li J-F, Norville JE, Aach J, McCormack M, Zhang D,

Bush J, et al. Multiplex and homologous recombination–mediated genome editing in *Arabidopsis* and *Nicotiana benthamiana* using guide RNA and Cas9. Nature Biotechnology. 2013;**31**(8):688

[11] Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S. Targeted mutagenesis in the model plant *Nicotiana benthamiana* using Cas9 RNA-guided endonuclease. Nature Biotechnology. 2013;**31**(8):691

[12] Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, et al. Targeted genome modification of crop plants using a CRISPR-Cas system. Nature Biotechnology. 2013;**31**(8):686

[13] Sun X, Hu Z, Chen R, Jiang Q, Song G, Zhang H, et al. Targeted mutagenesis in soybean using the CRISPR-Cas9 system. Scientific Reports. 2015;**5**:10342

[14] Jacobs TB, LaFayette PR, Schmitz RJ, Parrott WA. Targeted genome modifications in soybean with CRISPR/Cas9. BMC Biotechnology. 2015;**15**(1):16

[15] Cai Y, Chen L, Liu X, Sun S, Wu C, Jiang B, et al. CRISPR/Cas9-mediated genome editing in soybean hairy roots. PLoS One. 2015;**10**(8):e0136064

[16] Brooks C, Nekrasov V, Lippman ZB, Van Eck J. Efficient gene editing in tomato in the first generation using the clustered regularly interspaced short palindromic repeats/CRISPRassociated9 system. Plant Physiology. 2014;**166**(3):1292-1297

[17] Pan C, Ye L, Qin L, Liu X, He Y, Wang J, et al. CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations. Scientific Reports. 2016;**6**:24765

[18] Ito Y, Nishizawa-Yokoi A, Endo M, Mikami M, Toki S. CRISPR/Cas9 mediated mutagenesis of the RIN locus that regulates tomato fruit ripening. Biochemical and Biophysical Research Communications. 2015;**467**(1):76-82

[19] Ueta R, Abe C, Watanabe T, Sugano SS, Ishihara R, Ezura H, et al. Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9. Scientific Reports. 2017;**7**(1):507

[20] Wang S, Zhang S, Wang W, Xiong X, Meng F, Cui X. Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system. Plant Cell Reports. 2015;**34**(9):1473-1476

[21] Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C, Pearlsman M, et al. Development of broad virus resistance in non-transgenic cucumber using CRISPR/Cas9 technology. Molecular Plant Pathology. 2016;**17**(7):1140-1153

[22] Char SN, Neelakandan AK, Nahampun H, Frame B, Main M, Spalding MH, et al. An Agrobacteriumdelivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize. Plant Biotechnology Journal. 2017;**15**(2):257-268

[23] Svitashev S, Schwartz C, Lenderts B, Young JK, Cigan AM. Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nature Communications. 2016;**7**:13274

[24] Morineau C, Bellec Y, Tellier F, Gissot L, Kelemen Z, Nogué F, et al. Selective gene dosage by CRISPR-Cas9 genome editing in hexaploid *Camelina sativa*. Plant Biotechnology Journal. 2017;**15**(6):729-739

[25] Malnoy M, Viola R, Jung M-H, Koo O-J, Kim S, Kim J-S, et al. DNAfree genetically edited grapevine and apple protoplast using CRISPR/Cas9

ribonucleoproteins. Frontiers in Plant Science. 2016;**7**:1904

[26] Lawrenson T, Shorinola O, Stacey N, Li C, Østergaard L, Patron N, et al. Induction of targeted, heritable mutations in barley and *Brassica oleracea* using RNA-guided Cas9 nuclease. Genome Biology. 2015;**16**(1):258

[27] Tian S, Jiang L, Gao Q, Zhang J, Zong M, Zhang H, et al. Efficient CRISPR/Cas9-based gene knockout in watermelon. Plant Cell Reports. 2017;**36**(3):399-406

[28] Jia H, Wang N. Targeted genome editing of sweet orange using Cas9/ sgRNA. PLoS One. 2014;**9**(4):e93806

[29] Fan D, Liu T, Li C, Jiao B, Li S, Hou Y, et al. Efficient CRISPR/Cas9 mediated targeted mutagenesis in *Populus* in the first generation. Scientific Reports. 2015;**5**:12217

[30] Shi J, Gao H, Wang H, Lafitte HR, Archibald RL, Yang M, et al. ARGOS 8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnology Journal. 2017;**15**(2):207-216

[31] Zhou H, Liu B, Weeks DP, Spalding MH, Yang B. Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Research. 2014;**42**(17):10903-10914

[32] Endo M, Mikami M, Toki S. Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice. Plant and Cell Physiology. 2015;**56**(1):41-47

[33] Xu R-F, Li H, Qin R-Y, Li J, Qiu C-H, Yang Y-C, et al. Generation of inheritable and "transgene clean" targeted genome-modified rice in later generations using the CRISPR/Cas9 system. Scientific Reports. 2015;**5**:11491

**17**

2016;**7**:377

*CRISPR/Cas9-Mediated Gene Editing in Grain Crops DOI: http://dx.doi.org/10.5772/intechopen.88115*

> [42] Gil-Humanes J, Wang Y, Liang Z, Shan Q, Ozuna CV, Sánchez-León S, et al. High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. The Plant Journal.

[43] Xu R, Qin R, Li H, Li D, Li L, Wei P, et al. Generation of targeted mutant rice using a CRISPR-Cpf1 system. Plant Biotechnology Journal.

[44] Nandy S, Pathak B, Zhao S, Srivastava V. Heat-shock-inducible CRISPR/Cas9 system generates heritable mutations in rice. Plant Direct. 2019;**3**:1-14. DOI: 10.1002/

[45] Xu R, Yang Y, Qin R, Li H, Qiu C, Li L, et al. Rapid improvement of grain weight via highly efficient CRISPR/Cas9 mediated multiplex genome editing in rice. Journal of Genetics and Genomics =

Yi chuan xue bao. 2016;**43**(8):529

[46] Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, et al. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nature Communications. 2016;**7**:12617

[47] Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, et al. Efficient DNAfree genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nature Communications.

[48] Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable

resistance to powdery mildew. Nature

Biotechnology. 2014;**32**(9):947

[49] Chen R, Xu Q, Liu Y, Zhang J, Ren D, Wang G, et al. Generation of transgene-free maize male sterile lines using the CRISPR/Cas9 system. Frontiers in Plant Science. 2018;**9**:1180

2017;**89**(6):1251-1262

2017;**15**(6):713-717

pld3.145

2017;**8**:14261

[34] Lu Y, Zhu J-K. Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system. Molecular Plant. 2017;**10**(3):523-525

[35] Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Research. 2013;**41**(20):e188

[36] Xie K, Yang Y. RNA-guided genome editing in plants using a CRISPR–Cas system. Molecular Plant.

[37] Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, et al. Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS One.

[38] Sun Y, Zhang X, Wu C, He Y, Ma Y, Hou H, et al. Engineering herbicideresistant rice plants through CRISPR/

recombination of acetolactate synthase. Molecular Plant. 2016;**9**(4):628-631

[39] Li Q, Zhang D, Chen M, Liang W, Wei J, Qi Y, et al. Development of japonica photo-sensitive genic male sterile rice lines by editing carbon starved anther using CRISPR/Cas9. Journal of Genetics and Genomics = Yi

2013;**6**(6):1975-1983

2016;**11**(4):e0154027

Cas9-mediated homologous

chuan xue bao. 2016;**43**(6):415

[40] Zhou H, He M, Li J, Chen L, Huang Z, Zheng S, et al. Development of commercial thermo-sensitive genic male sterile rice accelerates hybrid rice breeding using the CRISPR/ Cas9-mediated TMS5 editing system. Scientific Reports. 2016;**6**:37395

[41] Li M, Li X, Zhou Z, Wu P, Fang M, Pan X, et al. Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system. Frontiers in Plant Science.

*CRISPR/Cas9-Mediated Gene Editing in Grain Crops DOI: http://dx.doi.org/10.5772/intechopen.88115*

[34] Lu Y, Zhu J-K. Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system. Molecular Plant. 2017;**10**(3):523-525

*Recent Advances in Grain Crops Research*

[19] Ueta R, Abe C, Watanabe T, Sugano SS, Ishihara R, Ezura H, et al. Rapid breeding of parthenocarpic tomato plants using CRISPR/Cas9. Scientific Reports. 2017;**7**(1):507

[20] Wang S, Zhang S, Wang W, Xiong X, Meng F, Cui X. Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system. Plant Cell Reports. 2015;**34**(9):1473-1476

[21] Chandrasekaran J, Brumin M, Wolf D, Leibman D, Klap C,

Pearlsman M, et al. Development of broad virus resistance in non-transgenic

technology. Molecular Plant Pathology.

[23] Svitashev S, Schwartz C, Lenderts B, Young JK, Cigan AM. Genome editing in maize directed by CRISPR–Cas9 ribonucleoprotein complexes. Nature Communications. 2016;**7**:13274

[24] Morineau C, Bellec Y, Tellier F, Gissot L, Kelemen Z, Nogué F, et al. Selective gene dosage by CRISPR-Cas9 genome editing in hexaploid *Camelina sativa*. Plant Biotechnology Journal.

[25] Malnoy M, Viola R, Jung M-H, Koo O-J, Kim S, Kim J-S, et al. DNAfree genetically edited grapevine and apple protoplast using CRISPR/Cas9

cucumber using CRISPR/Cas9

[22] Char SN, Neelakandan AK, Nahampun H, Frame B, Main M, Spalding MH, et al. An Agrobacteriumdelivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize. Plant Biotechnology Journal.

2016;**17**(7):1140-1153

2017;**15**(2):257-268

2017;**15**(6):729-739

[18] Ito Y, Nishizawa-Yokoi A, Endo M, Mikami M, Toki S. CRISPR/Cas9 mediated mutagenesis of the RIN locus that regulates tomato fruit ripening. Biochemical and Biophysical Research Communications. 2015;**467**(1):76-82

ribonucleoproteins. Frontiers in Plant

Stacey N, Li C, Østergaard L, Patron N, et al. Induction of targeted, heritable mutations in barley and *Brassica oleracea* using RNA-guided Cas9 nuclease. Genome Biology. 2015;**16**(1):258

[27] Tian S, Jiang L, Gao Q, Zhang J, Zong M, Zhang H, et al. Efficient CRISPR/Cas9-based gene knockout in watermelon. Plant Cell Reports.

[28] Jia H, Wang N. Targeted genome editing of sweet orange using Cas9/ sgRNA. PLoS One. 2014;**9**(4):e93806

[29] Fan D, Liu T, Li C, Jiao B, Li S, Hou Y, et al. Efficient CRISPR/Cas9 mediated targeted mutagenesis in *Populus* in the first generation. Scientific

[30] Shi J, Gao H, Wang H, Lafitte HR, Archibald RL, Yang M, et al. ARGOS 8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnology Journal.

[26] Lawrenson T, Shorinola O,

Science. 2016;**7**:1904

2017;**36**(3):399-406

Reports. 2015;**5**:12217

2017;**15**(2):207-216

2015;**56**(1):41-47

[31] Zhou H, Liu B, Weeks DP, Spalding MH, Yang B. Large

[32] Endo M, Mikami M, Toki S.

[33] Xu R-F, Li H, Qin R-Y, Li J, Qiu C-H, Yang Y-C, et al. Generation of inheritable and "transgene clean" targeted genome-modified rice in later generations using the CRISPR/Cas9 system. Scientific Reports. 2015;**5**:11491

Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice. Plant and Cell Physiology.

chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice. Nucleic Acids Research. 2014;**42**(17):10903-10914

**16**

[35] Jiang W, Zhou H, Bi H, Fromm M, Yang B, Weeks DP. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice. Nucleic Acids Research. 2013;**41**(20):e188

[36] Xie K, Yang Y. RNA-guided genome editing in plants using a CRISPR–Cas system. Molecular Plant. 2013;**6**(6):1975-1983

[37] Wang F, Wang C, Liu P, Lei C, Hao W, Gao Y, et al. Enhanced rice blast resistance by CRISPR/Cas9-targeted mutagenesis of the ERF transcription factor gene OsERF922. PLoS One. 2016;**11**(4):e0154027

[38] Sun Y, Zhang X, Wu C, He Y, Ma Y, Hou H, et al. Engineering herbicideresistant rice plants through CRISPR/ Cas9-mediated homologous recombination of acetolactate synthase. Molecular Plant. 2016;**9**(4):628-631

[39] Li Q, Zhang D, Chen M, Liang W, Wei J, Qi Y, et al. Development of japonica photo-sensitive genic male sterile rice lines by editing carbon starved anther using CRISPR/Cas9. Journal of Genetics and Genomics = Yi chuan xue bao. 2016;**43**(6):415

[40] Zhou H, He M, Li J, Chen L, Huang Z, Zheng S, et al. Development of commercial thermo-sensitive genic male sterile rice accelerates hybrid rice breeding using the CRISPR/ Cas9-mediated TMS5 editing system. Scientific Reports. 2016;**6**:37395

[41] Li M, Li X, Zhou Z, Wu P, Fang M, Pan X, et al. Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system. Frontiers in Plant Science. 2016;**7**:377

[42] Gil-Humanes J, Wang Y, Liang Z, Shan Q, Ozuna CV, Sánchez-León S, et al. High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. The Plant Journal. 2017;**89**(6):1251-1262

[43] Xu R, Qin R, Li H, Li D, Li L, Wei P, et al. Generation of targeted mutant rice using a CRISPR-Cpf1 system. Plant Biotechnology Journal. 2017;**15**(6):713-717

[44] Nandy S, Pathak B, Zhao S, Srivastava V. Heat-shock-inducible CRISPR/Cas9 system generates heritable mutations in rice. Plant Direct. 2019;**3**:1-14. DOI: 10.1002/ pld3.145

[45] Xu R, Yang Y, Qin R, Li H, Qiu C, Li L, et al. Rapid improvement of grain weight via highly efficient CRISPR/Cas9 mediated multiplex genome editing in rice. Journal of Genetics and Genomics = Yi chuan xue bao. 2016;**43**(8):529

[46] Zhang Y, Liang Z, Zong Y, Wang Y, Liu J, Chen K, et al. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA. Nature Communications. 2016;**7**:12617

[47] Liang Z, Chen K, Li T, Zhang Y, Wang Y, Zhao Q, et al. Efficient DNAfree genome editing of bread wheat using CRISPR/Cas9 ribonucleoprotein complexes. Nature Communications. 2017;**8**:14261

[48] Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, et al. Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology. 2014;**32**(9):947

[49] Chen R, Xu Q, Liu Y, Zhang J, Ren D, Wang G, et al. Generation of transgene-free maize male sterile lines using the CRISPR/Cas9 system. Frontiers in Plant Science. 2018;**9**:1180 [50] Sánchez-León S, Gil-Humanes J, Ozuna CV, Giménez MJ, Sousa C, Voytas DF, et al. Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9. Plant Biotechnology Journal. 2018;**16**(4):902-910

[51] Chilcoat D, Liu Z-B, Sander J. Chapter two—Use of CRISPR/Cas9 for crop improvement in maize and soybean. In: Weeks D, Yang B, editors. Progress in Molecular Biology and Translational Science. Vol. 149. USA: Academic Press; 26 Jun 2017. pp. 27-46. ISBN:<PHONE_NUMBER>439

[52] Svitashev S, Young JK, Schwartz C, Gao H, Falco SC, Cigan AM. Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiology. 2015;**169**(2):931-945

[53] Zhu T, Peterson DJ, Tagliani L, Clair GS, Baszczynski CL, Bowen B. Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides. Proceedings of the National Academy of Sciencesof the United States of America. 1999;**96**(15):8768-8773

[54] Terada R, Urawa H, Inagaki Y, Tsugane K, Iida S. Efficient gene targeting by homologous recombination in rice. Nature Biotechnology. 2002;**20**(10):1030

[55] Gao H, Smith J, Yang M, Jones S, Djukanovic V, Nicholson MG, et al. Heritable targeted mutagenesis in maize using a designed endonuclease. The Plant Journal. 2010;**61**(1):176-187

[56] Mrizova K, Holaskova E, Öz MT, Jiskrova E, Frebort I, Galuszka P. Transgenic barley: A prospective tool for biotechnology and agriculture. Biotechnology Advances. 2014;**32**(1):137-157

[57] Arazoe T, Miyoshi K, Yamato T, Ogawa T, Ohsato S, Arie T, et al. Tailormade CRISPR/Cas system for highly

efficient targeted gene replacement in the rice blast fungus. Biotechnology and Bioengineering. 2015;**112**:2543-2549

[58] Barman HN, Sheng Z, Fiaz S, Zhong M, Wu Y, Cai Y, et al. Generation of a new thermo-sensitive genic male sterile rice line by targeted mutagenesis of TMS5 gene through CRISPR/Cas9 system. BMC Plant Biology. 2019;**19**:109

[59] Minkenberg B, Xie K, Yang Y. Discovery of rice essential genes by characterizing a CRISPR-edited mutation of closely related rice MAP kinase genes. The Plant Journal. 2017;**89**:636-648

[60] Nieves-Cordones M, Mohamed S, Tanoi K, Kobayashi NI, Takagi K, Vernet A, et al. Production of low-Cs(+) rice plants by inactivation of the K(+) transporter OsHAK1 with the CRISPR-Cas system. The Plant Journal. 2017;**92**:43-56

[61] Jiang M, Hu H, Kai J, Traw MB, Yang S, Zhang X. Different knockout genotypes of OsIAA23 in rice using CRISPR/Cas9 generating different phenotypes. Plant Molecular Biology. 2019;**100**:467-479

[62] Li J, Meng X, Zong Y, Chen K, Zhang H, Liu J, et al. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9. Nature Plants. 2016;**2**:16139

[63] Perez L, Soto E, Farre G, Juanos J, Villorbina G, Bassie L, et al. CRISPR/ Cas9 mutations in the rice Waxy/ GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis. Plant Cell Reports. 2019;**38**:417-433

[64] Perez L, Soto E, Villorbina G, Bassie L, Medina V, Munoz P, et al. CRISPR/Cas9-induced monoallelic mutations in the cytosolic AGPase large subunit gene APL2 induce the ectopic expression of APL2 and the

**19**

*CRISPR/Cas9-Mediated Gene Editing in Grain Crops DOI: http://dx.doi.org/10.5772/intechopen.88115*

> male-sterile hexaploid wheat lines for use in hybrid seed production. Plant Biotechnology Journal. 2019. DOI:

[73] Singh M, Kumar M, Albertsen MC, Young JK, Cigan AM. Concurrent modifications in the three homeologs of Ms45 gene with CRISPR-Cas9 lead to rapid generation of male sterile bread wheat (*Triticum aestivu*m L.). Plant Molecular Biology. 2018;**97**:371-383

[74] Feng C, Su H, Bai H, Wang R, Liu Y, Guo X, et al. High-efficiency genome editing using a dmc1 promotercontrolled CRISPR/Cas9 system in maize. Plant Biotechnology Journal.

[75] Feng C, Yuan J, Wang R, Liu Y, Birchler JA, Han F. Efficient targeted genome modification in maize using CRISPR/Cas9 system. Journal of Genetics and Genomics. 2016;**43**:37-43

[76] Shi J, Gao H, Wang H, Lafitte HR, Archibald RL, Yang M, et al. ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnology Journal. 2017;**15**:207-216

Przyborowski M, Lyznik LA, Orczyk W, Nadolska-Orczyk A. A simple and efficient CRISPR/Cas9 platform for induction of single and multiple, heritable mutations in barley (*Hordeum vulgare* L.). Plant Methods. 2018;**14**:111

[78] Holme IB, Wendt T, Gil-Humanes J, Deleuran LC, Starker CG, Voytas DF, et al. Evaluation of the mature grain phytase candidate HvPAPhy\_a gene in barley (*Hordeum vulgare* L.) using CRISPR/Cas9 and TALENs. Plant Molecular Biology. 2017;**95**:111-121

[79] Kapusi E, Corcuera-Gomez M, Melnik S, Stoger E. Heritable genomic fragment deletions and small indels in the putative ENGase gene induced by

[77] Gasparis S, Kala M,

10.1111/pbi.13106

2018;**16**:1848-1857

corresponding small subunit gene APS2b in rice leaves. Transgenic Research. 2018;**27**:423-439

[65] Saika H, Mori A, Endo M, Toki S. Targeted deletion of rice

[66] Sun Y, Jiao G, Liu Z, Zhang X, Li J, Guo X, et al. Generation of highamylose rice through CRISPR/Cas9 mediated targeted mutagenesis of starch branching enzymes. Frontiers in Plant

[67] Takeda Y, Suzuki S, Tobimatsu Y, Osakabe K, Osakabe Y, Ragamustari SK,

5-HYDROXYLASE loss-of-function mutants generated with the CRISPR/ Cas9 system. The Plant Journal.

[68] Tang L, Mao B, Li Y, Lv Q, Zhang L, Chen C, et al. Knockout of OsNramp5 using the CRISPR/Cas9 system produces

low Cd-accumulating indica rice without compromising yield. Scientific

[69] Yin X, Anand A, Quick P,

Biology. 1917;**2019**:257-268

2018;**18**:31-41

[70] Kim D, Alptekin B, Budak H. CRISPR/Cas9 genome editing in wheat. Functional and Integrative Genomics.

[71] Bhowmik P, Ellison E, Polley B, Bollina V, Kulkarni M, Ghanbarnia K, et al. Targeted mutagenesis in wheat microspores using CRISPR/Cas9. Scientific Reports. 2018;**8**:6502

[72] Okada A, Arndell T, Borisjuk N, Sharma N, Watson-Haigh NS, Tucker EJ, et al. CRISPR/Cas9-mediated knockout of Ms1 enables the rapid generation of

Bandyopadhyay A. Editing a stomatal developmental gene in rice with CRISPR/Cpf1. Methods in Molecular

et al. Lignin characterization of rice CONIFERALDEHYDE

Science. 2017;**8**:298

2019;**97**:543-554

Reports. 2017;**7**:14438

retrotransposon Tos17 via CRISPR/Cas9. Plant Cell Reports. 2019;**38**:455-458

*CRISPR/Cas9-Mediated Gene Editing in Grain Crops DOI: http://dx.doi.org/10.5772/intechopen.88115*

corresponding small subunit gene APS2b in rice leaves. Transgenic Research. 2018;**27**:423-439

*Recent Advances in Grain Crops Research*

[50] Sánchez-León S, Gil-Humanes J, Ozuna CV, Giménez MJ, Sousa C, Voytas DF, et al. Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9. Plant Biotechnology Journal. 2018;**16**(4):902-910

efficient targeted gene replacement in the rice blast fungus. Biotechnology and Bioengineering. 2015;**112**:2543-2549

[58] Barman HN, Sheng Z, Fiaz S, Zhong M, Wu Y, Cai Y, et al. Generation of a new thermo-sensitive genic male sterile rice line by targeted mutagenesis of TMS5 gene through CRISPR/Cas9 system. BMC Plant Biology. 2019;**19**:109

[59] Minkenberg B, Xie K, Yang Y. Discovery of rice essential genes by characterizing a CRISPR-edited mutation of closely related rice MAP kinase genes. The Plant Journal.

[60] Nieves-Cordones M, Mohamed S, Tanoi K, Kobayashi NI, Takagi K, Vernet A, et al. Production of low-Cs(+)

rice plants by inactivation of the K(+) transporter OsHAK1 with the CRISPR-Cas system. The Plant Journal.

[61] Jiang M, Hu H, Kai J, Traw MB, Yang S, Zhang X. Different knockout genotypes of OsIAA23 in rice using CRISPR/Cas9 generating different phenotypes. Plant Molecular Biology.

[62] Li J, Meng X, Zong Y, Chen K, Zhang H, Liu J, et al. Gene replacements and insertions in rice by intron targeting using CRISPR-Cas9. Nature Plants.

[63] Perez L, Soto E, Farre G, Juanos J, Villorbina G, Bassie L, et al. CRISPR/ Cas9 mutations in the rice Waxy/ GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis. Plant

Cell Reports. 2019;**38**:417-433

[64] Perez L, Soto E, Villorbina G, Bassie L, Medina V, Munoz P, et al. CRISPR/Cas9-induced monoallelic mutations in the cytosolic AGPase large subunit gene APL2 induce the ectopic expression of APL2 and the

2017;**89**:636-648

2017;**92**:43-56

2019;**100**:467-479

2016;**2**:16139

[51] Chilcoat D, Liu Z-B, Sander J. Chapter two—Use of CRISPR/Cas9 for crop improvement in maize and soybean. In: Weeks D, Yang B, editors. Progress in Molecular Biology and Translational Science. Vol. 149. USA: Academic Press; 26 Jun 2017. pp. 27-46. ISBN:<PHONE_NUMBER>439

[52] Svitashev S, Young JK, Schwartz C, Gao H, Falco SC, Cigan AM. Targeted mutagenesis, precise gene editing, and site-specific gene insertion in maize using Cas9 and guide RNA. Plant Physiology. 2015;**169**(2):931-945

[53] Zhu T, Peterson DJ, Tagliani L,

Bowen B. Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides.

[54] Terada R, Urawa H, Inagaki Y, Tsugane K, Iida S. Efficient gene

in rice. Nature Biotechnology.

Proceedings of the National Academy of Sciencesof the United States of America.

targeting by homologous recombination

[55] Gao H, Smith J, Yang M, Jones S, Djukanovic V, Nicholson MG, et al. Heritable targeted mutagenesis in maize using a designed endonuclease. The Plant Journal. 2010;**61**(1):176-187

[56] Mrizova K, Holaskova E, Öz MT, Jiskrova E, Frebort I, Galuszka P. Transgenic barley: A prospective tool for biotechnology and agriculture.

[57] Arazoe T, Miyoshi K, Yamato T, Ogawa T, Ohsato S, Arie T, et al. Tailormade CRISPR/Cas system for highly

Biotechnology Advances. 2014;**32**(1):137-157

Clair GS, Baszczynski CL,

1999;**96**(15):8768-8773

2002;**20**(10):1030

**18**

[65] Saika H, Mori A, Endo M, Toki S. Targeted deletion of rice retrotransposon Tos17 via CRISPR/Cas9. Plant Cell Reports. 2019;**38**:455-458

[66] Sun Y, Jiao G, Liu Z, Zhang X, Li J, Guo X, et al. Generation of highamylose rice through CRISPR/Cas9 mediated targeted mutagenesis of starch branching enzymes. Frontiers in Plant Science. 2017;**8**:298

[67] Takeda Y, Suzuki S, Tobimatsu Y, Osakabe K, Osakabe Y, Ragamustari SK, et al. Lignin characterization of rice CONIFERALDEHYDE 5-HYDROXYLASE loss-of-function mutants generated with the CRISPR/ Cas9 system. The Plant Journal. 2019;**97**:543-554

[68] Tang L, Mao B, Li Y, Lv Q, Zhang L, Chen C, et al. Knockout of OsNramp5 using the CRISPR/Cas9 system produces low Cd-accumulating indica rice without compromising yield. Scientific Reports. 2017;**7**:14438

[69] Yin X, Anand A, Quick P, Bandyopadhyay A. Editing a stomatal developmental gene in rice with CRISPR/Cpf1. Methods in Molecular Biology. 1917;**2019**:257-268

[70] Kim D, Alptekin B, Budak H. CRISPR/Cas9 genome editing in wheat. Functional and Integrative Genomics. 2018;**18**:31-41

[71] Bhowmik P, Ellison E, Polley B, Bollina V, Kulkarni M, Ghanbarnia K, et al. Targeted mutagenesis in wheat microspores using CRISPR/Cas9. Scientific Reports. 2018;**8**:6502

[72] Okada A, Arndell T, Borisjuk N, Sharma N, Watson-Haigh NS, Tucker EJ, et al. CRISPR/Cas9-mediated knockout of Ms1 enables the rapid generation of

male-sterile hexaploid wheat lines for use in hybrid seed production. Plant Biotechnology Journal. 2019. DOI: 10.1111/pbi.13106

[73] Singh M, Kumar M, Albertsen MC, Young JK, Cigan AM. Concurrent modifications in the three homeologs of Ms45 gene with CRISPR-Cas9 lead to rapid generation of male sterile bread wheat (*Triticum aestivu*m L.). Plant Molecular Biology. 2018;**97**:371-383

[74] Feng C, Su H, Bai H, Wang R, Liu Y, Guo X, et al. High-efficiency genome editing using a dmc1 promotercontrolled CRISPR/Cas9 system in maize. Plant Biotechnology Journal. 2018;**16**:1848-1857

[75] Feng C, Yuan J, Wang R, Liu Y, Birchler JA, Han F. Efficient targeted genome modification in maize using CRISPR/Cas9 system. Journal of Genetics and Genomics. 2016;**43**:37-43

[76] Shi J, Gao H, Wang H, Lafitte HR, Archibald RL, Yang M, et al. ARGOS8 variants generated by CRISPR-Cas9 improve maize grain yield under field drought stress conditions. Plant Biotechnology Journal. 2017;**15**:207-216

[77] Gasparis S, Kala M, Przyborowski M, Lyznik LA, Orczyk W, Nadolska-Orczyk A. A simple and efficient CRISPR/Cas9 platform for induction of single and multiple, heritable mutations in barley (*Hordeum vulgare* L.). Plant Methods. 2018;**14**:111

[78] Holme IB, Wendt T, Gil-Humanes J, Deleuran LC, Starker CG, Voytas DF, et al. Evaluation of the mature grain phytase candidate HvPAPhy\_a gene in barley (*Hordeum vulgare* L.) using CRISPR/Cas9 and TALENs. Plant Molecular Biology. 2017;**95**:111-121

[79] Kapusi E, Corcuera-Gomez M, Melnik S, Stoger E. Heritable genomic fragment deletions and small indels in the putative ENGase gene induced by

CRISPR/Cas9 in barley. Frontiers in Plant Science. 2017;**8**:540

[80] Liu G, Li J, Godwin ID. Genome editing by CRISPR/Cas9 in sorghum through biolistic bombardment. Methods in Molecular Biology. 1931;**2019**:169-183

**21**

**Chapter 3**

**Abstract**

rust resistance

**1. Introduction**

Transgenics

*and Gyuhwa Chung*

and/or to study their molecular control.

Wheat in the Era of Genomics and

*Usman Babar, Usama Arshad, Muhammad Tehseen Azhar,*

Wheat, as one of the most important cereal crops in the world and second major caloric source in the world after rice, is the major staple food in South Asia and many other countries of the world. Prior to onset of "Green Revolution," South Asian countries were facing the threat of severe famine. Green Revolution wheat genotypes brought out these countries from the crisis they were facing and has helped them to sustain their productions for more than half a century. With the emergence of molecular biology and biotechnology, another window of opportunity is opened to sustain wheat yields by using modern techniques of genes identification and utilization. Through this chapter, we have tried to gather information that was generated for wheat improvement in last 3 decades. These afforest included the development of molecular markers, mapping of genes, sequencing of markers genes, and their utilization through marker-assisted selection. The other part recorded various efforts to genetically transform wheat for traits improvements

**Keywords:** wheat, marker-assisted selection (MAS), transformation, gene mapping,

Wheat (*Triticum aestivum*) belonging to *Triticeae* tribe of *Poaceae* family exhibits the most complex allohexaploid genome of approximately 17 Gb. Moreover, wheat is the second largest crop of the world after rice, but it has higher nutrition value than rice and is consumed by more than 2.5 billion people across the world. It is cultivated over a land of 215 million hectares worldwide every year, which is more than that of any other crop of commercial significance. Growing on a diverse range of environments and responding variably to temperate, tropical, and subtropical climates during spring and winter seasons, wheat is the most crucial as well as dominant staple food of Asia and North Africa. It is highly susceptible to abiotic stresses such as higher temperatures and depression in its yield could be resulted which is not permissible for a major food crop. Green revolution based upon a single objective to improve the yields of major cereal crops resulted in major yield shift for wheat during the previous

century [1, 2]. Global export value of wheat is around 50 billion US\$ [3–5].

*Rana Muhammad Atif, Abdulaziz Abdullah Alsahli,*

*Ibrahim A. Alaraidh, Aysha Kiran, Iqrar Ahmad Rana*

#### **Chapter 3**

*Recent Advances in Grain Crops Research*

CRISPR/Cas9 in barley. Frontiers in

[80] Liu G, Li J, Godwin ID. Genome editing by CRISPR/Cas9 in sorghum through biolistic bombardment. Methods in Molecular Biology.

Plant Science. 2017;**8**:540

1931;**2019**:169-183

**20**

## Wheat in the Era of Genomics and Transgenics

*Usman Babar, Usama Arshad, Muhammad Tehseen Azhar, Rana Muhammad Atif, Abdulaziz Abdullah Alsahli, Ibrahim A. Alaraidh, Aysha Kiran, Iqrar Ahmad Rana and Gyuhwa Chung*

#### **Abstract**

Wheat, as one of the most important cereal crops in the world and second major caloric source in the world after rice, is the major staple food in South Asia and many other countries of the world. Prior to onset of "Green Revolution," South Asian countries were facing the threat of severe famine. Green Revolution wheat genotypes brought out these countries from the crisis they were facing and has helped them to sustain their productions for more than half a century. With the emergence of molecular biology and biotechnology, another window of opportunity is opened to sustain wheat yields by using modern techniques of genes identification and utilization. Through this chapter, we have tried to gather information that was generated for wheat improvement in last 3 decades. These afforest included the development of molecular markers, mapping of genes, sequencing of markers genes, and their utilization through marker-assisted selection. The other part recorded various efforts to genetically transform wheat for traits improvements and/or to study their molecular control.

**Keywords:** wheat, marker-assisted selection (MAS), transformation, gene mapping, rust resistance

#### **1. Introduction**