**2. Brief description of induced mutation**

Mutation breeding carried out through three types of mutation as induced mutation, site-directed mutation and insertion mutation [1]. Induced mutation is a tool of generating variability artificially [18]. Mutagenesis is sudden and heritable changes in genetic sequence that stimulated by some mutagens like physical, chemical as well as biological agents [19]. Mutagenesis became well-known in 1950s when various crop species were largely induced through irradiation to enhance trait divergence [20]. Natural mutants are typical type of spontaneous mutations that generate modern phenotypes without human beings interference such as seed dispersal, thin seed coat, seed dormancy and reduced seed length. Heritable mutants are appropriate for human utilization for example loss of bitterness in various types of nuts, almonds, watermelons, potatoes, lima beans, egg plants as well as cabbages. Dwarfing genes exploited to increase grain yield in 1960s. It was completed with the introgression of natural mutant alleles into rice as well as wheat genome. The main disadvantage of this mutation is the loss of numerous wild features in crops [21].

Physical and chemical mutations are collectively called as induced mutation. Induced mutations have a record of 83 years as the first reported [22, 23] in plants. In 1927, Muller illustrated that X-ray induction could enhance the mutation rate in a *Drosophila* up to 15,000% [22] then Stadler examined a powerful phenotypic divergence in barley and maize by induction of X-rays and radium [23]. Induced mutations in plants originated directly from X-rays, radioactivity as well as radioactive elements through Roentgen (1895), Becquerel (1896) as well as Marie with Pierre Curie (1898) respectively. The Nobel Prize was awarded to Roentgen, Becquerel, Marie and Pierre Curie for successful mutation induction [24]. Nitrogen mustard is composed of poisonous mustard gas that applied in World Wars I and II. It is a chemical mutant that exposed to introduce mutations in cells [25]. Agents that cause artificial mutation are called mutagens that includes physical mutagens (X-ray, Gamma rays, Neutrons, Alpha/beta particles) and chemical mutagens (Alkylating agents, Azide, Hydroxylamine, Antibiotics, Nitrous acid) [1].

**11**

*Genetic Variability through Induced Mutation DOI: http://dx.doi.org/10.5772/intechopen.95027*

**2.1 Physical mutagens**

**2.2 Chemical mutagens**

American English is used consistently in your chapter.

Replace the entirety of this text with the main body of your chapter. The body is where the author explains experiments, presents and interprets data of one's research. Authors are free to decide how the main body will be structured. However, you are required to have at least one heading. Please ensure that either British or

Radiation is described as energy transfer in the sort of particles and waves [26]. These radiations are types of the electromagnetic (EM) spectrum that generates ions so it is also called as ionizing radiation. Ionizing radiations are the most frequently used physical mutagens [27]. Approximately, seventy percent of mutant varieties were generated by action of ionizing radiations in past eighty years [27]. These radiations consist of cosmic, gamma (γ) as well as X-rays [28]. Practical mutations by cosmic radiation have been reported in rice, cotton, wheat, tomato, sesame and pepper [29] as well as in maize [30]. The most universally applied physical mutagens are gamma and X-rays [27]. X-rays were the primary mutagens that applied to stimulate mutations [26]. However, gamma rays have gained popularity when these rays were accessible by the in several developing countries [31]. Gamma rays are less harmful produce point mutations with minute deletions while, fast neutron produces chromosome losses, translocations with huge deletions [32]. Additional physical agents are subatomic particles known as alpha (α), beta (β) particles, neutrons as well as protons. These particles are ionizing agents [26]. Ultraviolet (UV) rays are non-ionizing. These rays have potential of tissue penetrability for mutagenesis. Recently, plant materials have been thrown out into space for analysis of mutagenesis. Nevertheless,

information about genetics of space induction is so far insufficient [1].

powerfully carcinogenic that should be used carefully [37].

*2.2.1 Introduction and mode of action of ethyl methane sulfonate (EMS)*

Ethyl methane sulfonate is the most frequently applied in plants among chemical mutagens [37] due to its efficiency and accessibility [38]. It has capability to generate the high and stable nucleotide substitution in diverse genomes of organisms [39, 40].

Researchers search for another source for producing mutations due to the high chromosomal irregularity from ionizing emission. Consequently, a group of chemical induction has been exposed [33]. There are some chemical mutagens namely alkylating agents, base analogues as well as intercalating agents [25]. Alkylating agents were the primary group of chemical mutagens to be exposed by Auerbach and Robson [34] when they discovered the mutagenic result in mustard gas throughout World War II. Chemical mutagens consist of nitrogen mustards, sulfur mustards, ethyl methane sulphontes, ethyleneimines, epoxides, alkyl methane sulphonates, ethyleneimides, alky lnitrosoamines and alkyl nitrosoureas [35]. Chemical mutagens are more applicable for introduction of in-vitro mutation as compared to radiation approaches [36]. Chemical mutagens introduces single base pair (SNPs) change as compared to translocations and deletions as occurred in physical induction that induce more damage with harshly decrease viability. They are simple to apply rather than physical agents [32]. However, undesirable changes are usually high in chemical induction as compared to physical induction [26]. However, these mutagens are usually carcinogenic. Mustard gas, ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS) as well as nitrosoguanidine are

Replace the entirety of this text with the main body of your chapter. The body is where the author explains experiments, presents and interprets data of one's research. Authors are free to decide how the main body will be structured. However, you are required to have at least one heading. Please ensure that either British or American English is used consistently in your chapter.

### **2.1 Physical mutagens**

*Genetic Variation*

Spontaneous and Induced mutations are the primary source of all variations exists in an organisms that may be plants or animals [6]. Genetic variability endorses the differences among the same species and its existence in population is essential for its survival with changing environment. Induced mutation with specific mutagens generates the individuals with desirable characters that can be further exploit in breeding to generate new verities in plants [7, 8]. Mutation was first recognized in the late nineteenth century by Hugo de Varies, when he was working on the 'rediscovery' of Mendel's laws of inheritance [6]. Chemical mutagens are less harmful and easily available for work. In plants, widely used chemical mutagen is EMS that is very effective in causing point mutation in genome [9]. Mutation approaches produces huge and minute effect on all types of phenotypic traits [6]. Induced mutation is helpful in growing novel cultivars of plants as seedless grapes and edible bananas [3, 10] also it bring out the novel color variants of tuber and root crops [11]. Mutagenesis also apply to improve dwarfness, early growth, resistance to biotic and abiotic stresses,

and yield improvement as well as quality enhancements in plants [12–15].

targeted mutation permanently inside the genome [17].

**2. Brief description of induced mutation**

Novel breeding techniques based on biological mutagenic agent are widely introduce in plants for targeted variation is also known as targeted genome editing [16]. These genome editing techniques cause the specific and precise genome mutations. It introduce targeted mutation by either insertion, deletion that disturbs the function of gene. CRISPER/Cas9 is a novel technique that introduce desired

Mutation breeding carried out through three types of mutation as induced mutation, site-directed mutation and insertion mutation [1]. Induced mutation is a tool of generating variability artificially [18]. Mutagenesis is sudden and heritable changes in genetic sequence that stimulated by some mutagens like physical, chemical as well as biological agents [19]. Mutagenesis became well-known in 1950s when various crop species were largely induced through irradiation to enhance trait divergence [20]. Natural mutants are typical type of spontaneous mutations that generate modern phenotypes without human beings interference such as seed dispersal, thin seed coat, seed dormancy and reduced seed length. Heritable mutants are appropriate for human utilization for example loss of bitterness in various types of nuts, almonds, watermelons, potatoes, lima beans, egg plants as well as cabbages. Dwarfing genes exploited to increase grain yield in 1960s. It was completed with the introgression of natural mutant alleles into rice as well as wheat genome. The main disadvantage of this mutation is the loss of numerous wild features in crops [21]. Physical and chemical mutations are collectively called as induced mutation. Induced mutations have a record of 83 years as the first reported [22, 23] in plants. In 1927, Muller illustrated that X-ray induction could enhance the mutation rate in a *Drosophila* up to 15,000% [22] then Stadler examined a powerful phenotypic divergence in barley and maize by induction of X-rays and radium [23]. Induced mutations in plants originated directly from X-rays, radioactivity as well as radioactive elements through Roentgen (1895), Becquerel (1896) as well as Marie with Pierre Curie (1898) respectively. The Nobel Prize was awarded to Roentgen, Becquerel, Marie and Pierre Curie for successful mutation induction [24]. Nitrogen mustard is composed of poisonous mustard gas that applied in World Wars I and II. It is a chemical mutant that exposed to introduce mutations in cells [25]. Agents that cause artificial mutation are called mutagens that includes physical mutagens (X-ray, Gamma rays, Neutrons, Alpha/beta particles) and chemical mutagens (Alkylating agents, Azide, Hydroxylamine, Antibiotics, Nitrous acid) [1].

**10**

Radiation is described as energy transfer in the sort of particles and waves [26]. These radiations are types of the electromagnetic (EM) spectrum that generates ions so it is also called as ionizing radiation. Ionizing radiations are the most frequently used physical mutagens [27]. Approximately, seventy percent of mutant varieties were generated by action of ionizing radiations in past eighty years [27]. These radiations consist of cosmic, gamma (γ) as well as X-rays [28]. Practical mutations by cosmic radiation have been reported in rice, cotton, wheat, tomato, sesame and pepper [29] as well as in maize [30]. The most universally applied physical mutagens are gamma and X-rays [27]. X-rays were the primary mutagens that applied to stimulate mutations [26]. However, gamma rays have gained popularity when these rays were accessible by the in several developing countries [31]. Gamma rays are less harmful produce point mutations with minute deletions while, fast neutron produces chromosome losses, translocations with huge deletions [32]. Additional physical agents are subatomic particles known as alpha (α), beta (β) particles, neutrons as well as protons. These particles are ionizing agents [26]. Ultraviolet (UV) rays are non-ionizing. These rays have potential of tissue penetrability for mutagenesis. Recently, plant materials have been thrown out into space for analysis of mutagenesis. Nevertheless, information about genetics of space induction is so far insufficient [1].

### **2.2 Chemical mutagens**

Researchers search for another source for producing mutations due to the high chromosomal irregularity from ionizing emission. Consequently, a group of chemical induction has been exposed [33]. There are some chemical mutagens namely alkylating agents, base analogues as well as intercalating agents [25]. Alkylating agents were the primary group of chemical mutagens to be exposed by Auerbach and Robson [34] when they discovered the mutagenic result in mustard gas throughout World War II. Chemical mutagens consist of nitrogen mustards, sulfur mustards, ethyl methane sulphontes, ethyleneimines, epoxides, alkyl methane sulphonates, ethyleneimides, alky lnitrosoamines and alkyl nitrosoureas [35]. Chemical mutagens are more applicable for introduction of in-vitro mutation as compared to radiation approaches [36]. Chemical mutagens introduces single base pair (SNPs) change as compared to translocations and deletions as occurred in physical induction that induce more damage with harshly decrease viability. They are simple to apply rather than physical agents [32]. However, undesirable changes are usually high in chemical induction as compared to physical induction [26]. However, these mutagens are usually carcinogenic. Mustard gas, ethyl methane sulfonate (EMS), methyl methane sulfonate (MMS) as well as nitrosoguanidine are powerfully carcinogenic that should be used carefully [37].

#### *2.2.1 Introduction and mode of action of ethyl methane sulfonate (EMS)*

Ethyl methane sulfonate is the most frequently applied in plants among chemical mutagens [37] due to its efficiency and accessibility [38]. It has capability to generate the high and stable nucleotide substitution in diverse genomes of organisms [39, 40]. This chemical generates a huge quantity of point mutations in relatively little mutant population. This chemical is enough to develop the genome mutations [40]. EMS has major role in forward genetic for screening of various organisms. It is also applied in model animal and plant for mutagenesis named *Drosophila melanogaster* as well as *Arabidopsis thaliana* respectively. EMS is extraordinarily reliable due to similar levels of induction have been attained in model organisms for example base replacement are analogous for *Arabidopsis* seeds immersed in EMS [41, 42] as well as EMS-fed males *Drosophila* [43]. EMS causes suitable levels of lethality as well as sterility [40]. Genome size does not show to be a significant issue in EMS mutagenesis. Nevertheless, EMS toxicity may differ from species to species [44]. It is also applied in high throughput selection such as TILLING populations [37] in plants.

Ethyl methane sulfonate forms an abnormal base of O−6-ethyl guanine due to alkylation of guanine bases. During DNA duplication, it located a thymine residue above a cytosine residue result in an accidental point mutation. Approximately 70–99% alterations in EMS-treated populations are due to GC → AT base pair conversion [37, 40].

#### *2.2.2 Dose of mutagen*

LD50 is the percentage of test material that are killed by a specific dosage of chemical or radiation mutagen in which half test material will be die. Fixation of LD50 is important before the start of an experiment in induced mutation. These doses vary according to fluctuation in treatments duration, quantity, pH as well as solvent used. Mutagen dose can be caused low or high mutation frequency as a result of ignoring the importance of LD50 [45–47]. Doses lower than LD50 favor plant's recovery after treatment, while the use of high doses increases the probability to induce mutation either in positive or in negative direction. The efficiency of mutation is determined by concluding the accurate doses of mutagens if the dose is random it creates higher number of harmful mutations in each plant [48]. The mutation quality of practical mutation is not absolutely correlated to dose rate. High mutagen doses did not produced the excellent results of yield [26]. Seeds of *Oryza sativa* L. spp. *Indica* cv. MR219 were mutagenized to different doses of EMS from 0.25–2%. Seeds were incubated for ten to twenty hours for establishment of kill curve as well as sensitivity of the tested genotypes [49].

#### **2.3 Mutagenesis with biological agents**

Insertion mutageneis with biological agents involved in insertion or deletion of some sequence in genome. It may cause random mutation at genome as transfer DNA, retro-transposon and transposon. Also the insertion mutations can be site specific or targeted that cause genomic variation at specific site included the novel genome editing techniques.

Transfer DNA (T-DNA) insertion mutation helps in identification of gene function in plant genome. This insertion mutagenesis cause loss or gain of gene function that can be observed by phenotypic response [50]. It can also identified through whole genome sequencing or using the *Agrobacterium tumefaciens* machinery that insert the T-DNA at specific flanking sites.

#### *2.3.1 Mutation by genome editing tools*

Plant breeding relies on incorporation of genetic variation for desired traits. The innovative strategies are exploited from many years to reduce the off-target random mutations caused by physical and chemical mutagens [16]. These technologies

**13**

*Genetic Variability through Induced Mutation DOI: http://dx.doi.org/10.5772/intechopen.95027*

cause genetic variation [54].

ery enlisted in **Table 1**.

*BnaMAX1s* Gateway 100

*OsRR22* pYLCRISPR/

*MaGA20ox2* pYLCRISPR/

vectors; BGK01 vector: 35S promoter

pTF101.1: 35S promoter

Vas9Pubi-H; Cas9- OsRR22-gRNA: OsU6 promoter

Cas9Pubi-H vector: U3 promoter

pBIN-sgR-Cas9- OsU3 vector: 35S PROMOTER

pCas9-TPC: pMP90RK

**Gene targeted**

*GmFT2a; G,FT5a*

Exon of *SD1* gene

*BnSFAR4; BnSFAR5*

includes: site-directed nucleases, RNA-dependent methylation, oligonucleotide directed mutagenesis, agro infiltration, cisgenec/intragenic and reverse genetics. Site-directed nucleases including Zinc finger nucleases (ZFN) [51], transcription activator-like effector nucleases (TALEN) [52] and Clustered regularly interspaced short palindromic repeats (CRISPER) and CRISPER-associated nuclease 9 (CRISPER/Cas9) [53] system had revolutionized the mutation breeding strategy by introducing targeted genome editing. TALEN and CRISPER/Cas techniques precisely and permanently incorporate the desired DNA into the genome and hence

CRISPR system is a well settle defense system that generate acquired immune response for resistance to bacteria, fungi and phages [55]. It was originated from bacteria and consist of repetitive DNA genetic codes as well as proto-spacer DNA (defensive genetic codes formed during exposure to pathogens) [56, 57]. Cas9 is an endonuclease that generate double stranded break in DNA through its two active domains [58]. Cas9 creates the break at targeted site by utilizing the guided RNA sequence. The CRISPER/Cas system recognized the specific site at genome through guide RNA (gRNA) [59] and at targeted site Cas9 creates a double stranded break (DSB). These break are repaired by DNA repairing system that ultimately cause mutation by either non-homologous end joining (NHEJ) or homologous recombination system [HR] [60, 61]. Specific base-pair change occur during DNA repair system as Cytosine to thymine (C/T) [62] and Adenine to guanine (A/G) [63] that observed in several crop plants as canola, rice, tomato, wheat and corn [57]. CRISPR/Cas9 system was used in rice to generate semi-dwarf mutants in rice from T2 to T4 generation. Stable indels passed through generation producing homozygous mutant [64]. In plants, CRISPR/Cas system generates induced mutation through gene knockouts, insertion or generating single nucleotide polymorphism (SNP) in plants [56, 57]. Some latest gene editing mutations using CRISPR machin-

**Vector: promoter Transformation** 

**method: promoter**

Agrobacterium GV3101

Agrobacterium strain EHA101

Agrobacterium EHA105: OsU6

Agrobacterium strain EHA105

Agrobacterium strain LBA4404, EHA105

Agrobacterium GV3101

**Plant variety Mutation** 

Rapeseed RS862

Soybean jack variety (*Glycine max*)

Rice japonica WPB106

Banana cultivar Gro Michel) (Musa acuminate)

Rice variety 9815B, JIAODA138, HUAIDAO1055

Rapeseed RS306

**nature**

Knockout mutation

Knockout by CRISPR

Knockout mutation

Mutation as insertion and deletion

On target and off target mutations

Knockout mutation

**Reference**

[65]

[66]

[67]

[68]

[64]

[69]

#### *Genetic Variability through Induced Mutation DOI: http://dx.doi.org/10.5772/intechopen.95027*

*Genetic Variation*

conversion [37, 40].

*2.2.2 Dose of mutagen*

This chemical generates a huge quantity of point mutations in relatively little mutant population. This chemical is enough to develop the genome mutations [40]. EMS has major role in forward genetic for screening of various organisms. It is also applied in model animal and plant for mutagenesis named *Drosophila melanogaster* as well as *Arabidopsis thaliana* respectively. EMS is extraordinarily reliable due to similar levels of induction have been attained in model organisms for example base replacement are analogous for *Arabidopsis* seeds immersed in EMS [41, 42] as well as EMS-fed males *Drosophila* [43]. EMS causes suitable levels of lethality as well as sterility [40]. Genome size does not show to be a significant issue in EMS mutagenesis. Nevertheless, EMS toxicity may differ from species to species [44]. It is also applied

Ethyl methane sulfonate forms an abnormal base of O−6-ethyl guanine due to alkylation of guanine bases. During DNA duplication, it located a thymine residue above a cytosine residue result in an accidental point mutation. Approximately 70–99% alterations in EMS-treated populations are due to GC → AT base pair

LD50 is the percentage of test material that are killed by a specific dosage of chemical or radiation mutagen in which half test material will be die. Fixation of LD50 is important before the start of an experiment in induced mutation. These doses vary according to fluctuation in treatments duration, quantity, pH as well as solvent used. Mutagen dose can be caused low or high mutation frequency as a result of ignoring the importance of LD50 [45–47]. Doses lower than LD50 favor plant's recovery after treatment, while the use of high doses increases the probability to induce mutation either in positive or in negative direction. The efficiency of mutation is determined by concluding the accurate doses of mutagens if the dose is random it creates higher number of harmful mutations in each plant [48]. The mutation quality of practical mutation is not absolutely correlated to dose rate. High mutagen doses did not produced the excellent results of yield [26]. Seeds of *Oryza sativa* L. spp. *Indica* cv. MR219 were mutagenized to different doses of EMS from 0.25–2%. Seeds were incubated for ten to twenty hours for establishment of kill

Insertion mutageneis with biological agents involved in insertion or deletion of some sequence in genome. It may cause random mutation at genome as transfer DNA, retro-transposon and transposon. Also the insertion mutations can be site specific or targeted that cause genomic variation at specific site included the novel

Transfer DNA (T-DNA) insertion mutation helps in identification of gene function in plant genome. This insertion mutagenesis cause loss or gain of gene function that can be observed by phenotypic response [50]. It can also identified through whole genome sequencing or using the *Agrobacterium tumefaciens* machinery that

Plant breeding relies on incorporation of genetic variation for desired traits. The innovative strategies are exploited from many years to reduce the off-target random mutations caused by physical and chemical mutagens [16]. These technologies

curve as well as sensitivity of the tested genotypes [49].

**2.3 Mutagenesis with biological agents**

insert the T-DNA at specific flanking sites.

*2.3.1 Mutation by genome editing tools*

genome editing techniques.

in high throughput selection such as TILLING populations [37] in plants.

**12**

includes: site-directed nucleases, RNA-dependent methylation, oligonucleotide directed mutagenesis, agro infiltration, cisgenec/intragenic and reverse genetics. Site-directed nucleases including Zinc finger nucleases (ZFN) [51], transcription activator-like effector nucleases (TALEN) [52] and Clustered regularly interspaced short palindromic repeats (CRISPER) and CRISPER-associated nuclease 9 (CRISPER/Cas9) [53] system had revolutionized the mutation breeding strategy by introducing targeted genome editing. TALEN and CRISPER/Cas techniques precisely and permanently incorporate the desired DNA into the genome and hence cause genetic variation [54].

CRISPR system is a well settle defense system that generate acquired immune response for resistance to bacteria, fungi and phages [55]. It was originated from bacteria and consist of repetitive DNA genetic codes as well as proto-spacer DNA (defensive genetic codes formed during exposure to pathogens) [56, 57]. Cas9 is an endonuclease that generate double stranded break in DNA through its two active domains [58]. Cas9 creates the break at targeted site by utilizing the guided RNA sequence. The CRISPER/Cas system recognized the specific site at genome through guide RNA (gRNA) [59] and at targeted site Cas9 creates a double stranded break (DSB). These break are repaired by DNA repairing system that ultimately cause mutation by either non-homologous end joining (NHEJ) or homologous recombination system [HR] [60, 61]. Specific base-pair change occur during DNA repair system as Cytosine to thymine (C/T) [62] and Adenine to guanine (A/G) [63] that observed in several crop plants as canola, rice, tomato, wheat and corn [57]. CRISPR/Cas9 system was used in rice to generate semi-dwarf mutants in rice from T2 to T4 generation. Stable indels passed through generation producing homozygous mutant [64]. In plants, CRISPR/Cas system generates induced mutation through gene knockouts, insertion or generating single nucleotide polymorphism (SNP) in plants [56, 57]. Some latest gene editing mutations using CRISPR machinery enlisted in **Table 1**.



#### **Table 1.**

*Induced mutation in plants using targeted genome editing method CRISPR/Cas9.*
