**3. Mutation concept and its importance**

The word 'mutation' was coined by Hugo de Vries (1901) to represent a sudden heritable change occurring in the DNA of an organism caused artificially through irradiation, chemicals, viruses, transposons, or chromosomal aberrations that occur during reproductive processes [19]. These changes can be transferred to the offspring and are e differentiated in three general types namely gene mutation, chromosomal and genomic mutations. Induced mutation became the most frequently employed technique for developing novel improved germplasm in crop plants [20]. Mutation breeding is the application of mutagens to plant cells to accomplish crop breeding. Genetic variation makes the basis for the evolutionary process and breeding. In 1940, mutagenesis was adopted by the breeders as a tool that works faster to create mutations in plants [21]. Induced mutation breeding techniques have become most efficient, fast-tracking and widely exploited tools for crop improvement worldwide (**Figure 1**).

Mutation can be differentiated in three general types namely gene mutation, chromosomal and genomic mutations. However, mutation breeding is the application of mutagens to plant cells to accomplish crop breeding. Mutation provides the fundamental basis for a genetic variation on which genetic advancement and genetic drift depend and a single base mutation can cause devastating or beneficial consequences or no effect at all. Mutation breeding has played a significant role in crop breeding and genetics and genomic studies by generating a large amount of genetic diversity. Concurrently, climatic changes also threatening the food supply chain on the global level, resulting in fast loss of biodiversity for food and agriculture. The ongoing unpredictable climatic changes are the core problem in reducing crop yields worldwide, thus continuous development of new improved varieties for sustainable production is unavoidable. While the rate of natural mutations in the

#### **Figure 1.**

*Number of mutant varieties released in top 20 countries. Source: Mutant varieties database, IAEA accessed on 10th September, 2020.*

crop plants is rare, thus use of induced mutation is indispensable to create genetic diversity for the desired traits for use in the breeding programs. Developing a new variety through mutation breeding reduces the time span for varietal development as compared to hybridization (**Figure 2**). Moreover, Mutants with multiple traits can be discriminated through mutation breeding, mutant varieties show a higher survival rate in the face of environmental swings. Mutagenesis is an efficient tool for generating mutations; these mutations can occur naturally or can be induced using mutagens, broadly classified as physical and chemical mutagens [22]. Mutagens offer more chances to acquire desired phenotypic changes and to study the genetic variations in relation to phenotypes and the annotation /deciphering of gene functions [23]. Various genetic resources of crop plants have been developed globally using different mutagenesis sources like EMS, gamma or X-rays and fast neutrons [24]. The crops like tomato have been focused after the availability of whole-genome sequencing data, which led to the identification of millions of single nucleotide polymorphisms (SNPs) and indels in tomato lines and in mutants [23]. In view of the introduction of high throughput next-generations equencing (NGS), several innovative approaches have been introduced for the discrimination of mutations in the mutagenized material. Some remarkable techniques are MutMap (mapping-by-sequencing) and MutChromSeq helpful to identify the basic changes induced through mutagenesis [25]. MutChromSeq helps to assort the desired genes in the shortest time span and has been successfully utilized in wheat and barley. Pakistan Atomic Energy Commission's (PAEC) first agriculture institute, Nuclear Institute of Agriculture (NIA)Tandojam" has exploited mutation breeding techniques since its inception in 1963 and developed 3 mutant varieties of wheat, 7 of rice, 1 of sugarcane, 5 of cotton, one each of lentil, mungbean and rapeseed through mutation breeding techniques. NIA released the first rice variety (Shadab) in 1978 from IR6 using ethyl methane sulphonate (EMS 0.5%) a chemical mutagen, variety had the potential to produce 7 tones/ha with superior grain quality [26]. However, the Nuclear Institute for Agriculture and Biology (NIAB) and other institutes of

**89**

*Potential of Mutation Breeding to Sustain Food Security DOI: http://dx.doi.org/10.5772/intechopen.94087*

and boosting up their socio-economic status.

ness phenomenon in diploid organisms in 1866 [30].

**3.1 Spontaneous mutations**

*Scheme of mutation breeding in crop plants.*

**Figure 2.**

**3.2 Induced mutations**

PAEC have also developed mutant varieties of cotton, castor bean, sesame and mandarin thus helping the farming community by developing these improved varieties

These are the genetic changes that occur due to chromosomal aberrations in the biological processes and serves as raw material for the evolutionary process. These mutations are the alleles of unknown genes which afterward given the name according to the phenotype or other related information viz., super-root (surl-7 to surl-7) [27], maize bronze (bz), carbohydrate accumulation mutant (caml) [28]. In maize spontaneous mutations occur in high frequency in the pollen part of some maize genotypes, but not in others [29]. Recessive mutations (one or two copies of the mutated allele produces the phenotype) are denoted by small letters, whilst dominant (one or two copies of the mutated allele produces the phenotype) and partially-dominant (one mutant allele produces an intermediate phenotype) are denoted by the first letter capital followed by the small letters. Most of the spontaneous mutations are point (single base pair change in the DNA) mutations. Gregor John Mendel was the first to quantitatively evaluate the dominance and recessive-

In addition to naturally occurring genetic mutations, novel alleles have been induced in plants by chemical and physical mutagenesis (**Figure 3**). The goal of mutagenesis is to induce genetic variation in cells that give rise to plants while minimizing chimeras, sterility and lethality [31]. Mutagenesis based breeding is primarily used to improve 1 to 2 main traits that effect on productivity or quality traits. More importantly is not under the regulatory restrictions faced by the genetically

*Potential of Mutation Breeding to Sustain Food Security DOI: http://dx.doi.org/10.5772/intechopen.94087*

*Genetic Variation*

**Figure 1.**

*10th September, 2020.*

crop plants is rare, thus use of induced mutation is indispensable to create genetic diversity for the desired traits for use in the breeding programs. Developing a new variety through mutation breeding reduces the time span for varietal development as compared to hybridization (**Figure 2**). Moreover, Mutants with multiple traits can be discriminated through mutation breeding, mutant varieties show a higher survival rate in the face of environmental swings. Mutagenesis is an efficient tool for generating mutations; these mutations can occur naturally or can be induced using mutagens, broadly classified as physical and chemical mutagens [22]. Mutagens offer more chances to acquire desired phenotypic changes and to study the genetic variations in relation to phenotypes and the annotation /deciphering of gene functions [23]. Various genetic resources of crop plants have been developed globally using different mutagenesis sources like EMS, gamma or X-rays and fast neutrons [24]. The crops like tomato have been focused after the availability of whole-genome sequencing data, which led to the identification of millions of single nucleotide polymorphisms (SNPs) and indels in tomato lines and in mutants [23]. In view of the introduction of high throughput next-generations equencing (NGS), several innovative approaches have been introduced for the discrimination of mutations in the mutagenized material. Some remarkable techniques are MutMap (mapping-by-sequencing) and MutChromSeq helpful to identify the basic changes induced through mutagenesis [25]. MutChromSeq helps to assort the desired genes in the shortest time span and has been successfully utilized in wheat and barley. Pakistan Atomic Energy Commission's (PAEC) first agriculture institute, Nuclear Institute of Agriculture (NIA)Tandojam" has exploited mutation breeding techniques since its inception in 1963 and developed 3 mutant varieties of wheat, 7 of rice, 1 of sugarcane, 5 of cotton, one each of lentil, mungbean and rapeseed through mutation breeding techniques. NIA released the first rice variety (Shadab) in 1978 from IR6 using ethyl methane sulphonate (EMS 0.5%) a chemical mutagen, variety had the potential to produce 7 tones/ha with superior grain quality [26]. However, the Nuclear Institute for Agriculture and Biology (NIAB) and other institutes of

*Number of mutant varieties released in top 20 countries. Source: Mutant varieties database, IAEA accessed on* 

**88**

**Figure 2.** *Scheme of mutation breeding in crop plants.*

PAEC have also developed mutant varieties of cotton, castor bean, sesame and mandarin thus helping the farming community by developing these improved varieties and boosting up their socio-economic status.

## **3.1 Spontaneous mutations**

These are the genetic changes that occur due to chromosomal aberrations in the biological processes and serves as raw material for the evolutionary process. These mutations are the alleles of unknown genes which afterward given the name according to the phenotype or other related information viz., super-root (surl-7 to surl-7) [27], maize bronze (bz), carbohydrate accumulation mutant (caml) [28]. In maize spontaneous mutations occur in high frequency in the pollen part of some maize genotypes, but not in others [29]. Recessive mutations (one or two copies of the mutated allele produces the phenotype) are denoted by small letters, whilst dominant (one or two copies of the mutated allele produces the phenotype) and partially-dominant (one mutant allele produces an intermediate phenotype) are denoted by the first letter capital followed by the small letters. Most of the spontaneous mutations are point (single base pair change in the DNA) mutations. Gregor John Mendel was the first to quantitatively evaluate the dominance and recessiveness phenomenon in diploid organisms in 1866 [30].

### **3.2 Induced mutations**

In addition to naturally occurring genetic mutations, novel alleles have been induced in plants by chemical and physical mutagenesis (**Figure 3**). The goal of mutagenesis is to induce genetic variation in cells that give rise to plants while minimizing chimeras, sterility and lethality [31]. Mutagenesis based breeding is primarily used to improve 1 to 2 main traits that effect on productivity or quality traits. More importantly is not under the regulatory restrictions faced by the genetically

#### **Figure 3.**

*Common mutagens used in plant mutation breeding. Source: Reproduced from FAO/IAEA, 2018.*

modified organisms [32]. In some crops, chemically induced mutagenesis produced the desired phenotype in only several thousand lines. Today's high throughput phenotyping and next-generation sequencing methods have expedited the process to identify the mutants with desired genes (**Figure 4**). The use of engineered nucleases has helped to increase the accuracy of the mutation breeding through gene-specific mutation. Allelomorphic diversity induced in the gene of interest, whether spontaneously or experimentally, can be a great source for breeding programs to inculcate novel agricultural attributes [31]. Wanga et al. [33] used a combination of EMS and gamma radiation in sorghum but results were not recommendable. Although these are two major mutagens used to develop mutations [34, 35].

#### *3.2.1 Physical mutagenesis*

Physical mutagens namely X-rays, neutrons-alpha-beta particles, fast and thermal neutron, UV-light, especially gamma rays are used for the induction of mutation [36]. Physical mutagens are more common as compared to chemical mutagens (EMS) for mutagenesis. Physical mutagens like x-rays and gamma rays are preferred by the breeders as compared to the chemical ones. Gamma rays were used more frequently which accounted to improve 1604 mutants than the X-rays which improved 561 mutants [36]. Plant's exposure to X-rays provided the first ever undeniable evidence that phenotypic variability can be induced artificially. Hermann J. Muller was awarded Nobel Prize in 1946 in medicine/physiology for introducing irradiation using X-rays. Gamma-irradiation produces severe genetic mutations due to large chromosomal deletions and the re-enactment of the chromosome. Gamma rays have been used to induce mutations in seeds, cuttings, pollens and calli [37]. Since 1960 gamma irradiation has become the most popular and commonly used mutagens. This radiation-based mutagenesis was broadly used to improve mutant varieties directly as compared to other methods (acclimatization, selection, hybridization), comparatively, time-consuming, laborious and with

**91**

*Potential of Mutation Breeding to Sustain Food Security DOI: http://dx.doi.org/10.5772/intechopen.94087*

lower genetic variation [38]. Fast neutron-induced mutagenesis is an exceptional technique among the other mutagenesis tools being employed in crop science in relation to higher impact. Fast neutrons normally cause deletions from a small number of bases to million bases [39]. Although, previously fast neutron was not as

*Mutation breeding integrated use with modern techniques. Source: Directly taken from Jo and Kim, 2019.*

Space-induced mutation breeding uses cosmic rays to induce seeds in the space, for this experiment it is carried out in the satellites, space shuttles, and high altitude balloons and are considered beneficial over gamma radiation because of its lower damage to plants as compared to gamma rays on earth. Using space induced radiation, several advantageous mutations to make a breakthrough in yield were also achieved [13, 15, 41]. China has produced 41 varieties developed through space– induced mutation breeding of various crop species viz., rice, wheat, cotton, sesame,

Heavy-ion bean is an important tool in mutation breeding since lower radiation doses are found to induce high mutation rates [43]. Due to its dense localized effect on DNA to effectively alter a single trait of the irradiated cultivar without damaging the rest of the characteristics, this technique is effectively being used in China and Japan to produce a large number of mutant varieties [44, 45]. In Japan, several ornamental plant varieties have been developed using high-energy ion beam irradiation

popular as other physical mutagens in plant mutagenesis [40].

*3.2.2 Space mutagenesis*

**Figure 4.**

pepper, tomato, and alfalfa [42].

*3.2.3 Ion beam mutagenesis*

*Potential of Mutation Breeding to Sustain Food Security DOI: http://dx.doi.org/10.5772/intechopen.94087*

**Figure 4.**

*Genetic Variation*

modified organisms [32]. In some crops, chemically induced mutagenesis produced the desired phenotype in only several thousand lines. Today's high throughput phenotyping and next-generation sequencing methods have expedited the process to identify the mutants with desired genes (**Figure 4**). The use of engineered nucleases has helped to increase the accuracy of the mutation breeding through gene-specific mutation. Allelomorphic diversity induced in the gene of interest, whether spontaneously or experimentally, can be a great source for breeding programs to inculcate novel agricultural attributes [31]. Wanga et al. [33] used a combination of EMS and gamma radiation in sorghum but results were not recommendable. Although these

*Common mutagens used in plant mutation breeding. Source: Reproduced from FAO/IAEA, 2018.*

Physical mutagens namely X-rays, neutrons-alpha-beta particles, fast and thermal neutron, UV-light, especially gamma rays are used for the induction of mutation [36]. Physical mutagens are more common as compared to chemical mutagens (EMS) for mutagenesis. Physical mutagens like x-rays and gamma rays are preferred by the breeders as compared to the chemical ones. Gamma rays were used more frequently which accounted to improve 1604 mutants than the X-rays which improved 561 mutants [36]. Plant's exposure to X-rays provided the first ever undeniable evidence that phenotypic variability can be induced artificially. Hermann J. Muller was awarded Nobel Prize in 1946 in medicine/physiology for introducing irradiation using X-rays. Gamma-irradiation produces severe genetic mutations due to large chromosomal deletions and the re-enactment of the chromosome. Gamma rays have been used to induce mutations in seeds, cuttings, pollens and calli [37]. Since 1960 gamma irradiation has become the most popular and commonly used mutagens. This radiation-based mutagenesis was broadly used to improve mutant varieties directly as compared to other methods (acclimatization, selection, hybridization), comparatively, time-consuming, laborious and with

are two major mutagens used to develop mutations [34, 35].

*3.2.1 Physical mutagenesis*

**Figure 3.**

**90**

*Mutation breeding integrated use with modern techniques. Source: Directly taken from Jo and Kim, 2019.*

lower genetic variation [38]. Fast neutron-induced mutagenesis is an exceptional technique among the other mutagenesis tools being employed in crop science in relation to higher impact. Fast neutrons normally cause deletions from a small number of bases to million bases [39]. Although, previously fast neutron was not as popular as other physical mutagens in plant mutagenesis [40].

#### *3.2.2 Space mutagenesis*

Space-induced mutation breeding uses cosmic rays to induce seeds in the space, for this experiment it is carried out in the satellites, space shuttles, and high altitude balloons and are considered beneficial over gamma radiation because of its lower damage to plants as compared to gamma rays on earth. Using space induced radiation, several advantageous mutations to make a breakthrough in yield were also achieved [13, 15, 41]. China has produced 41 varieties developed through space– induced mutation breeding of various crop species viz., rice, wheat, cotton, sesame, pepper, tomato, and alfalfa [42].

#### *3.2.3 Ion beam mutagenesis*

Heavy-ion bean is an important tool in mutation breeding since lower radiation doses are found to induce high mutation rates [43]. Due to its dense localized effect on DNA to effectively alter a single trait of the irradiated cultivar without damaging the rest of the characteristics, this technique is effectively being used in China and Japan to produce a large number of mutant varieties [44, 45]. In Japan, several ornamental plant varieties have been developed using high-energy ion beam irradiation

while China is using low-energy Ion bean to create improved crop varieties. The initial plant varieties produced using Ion bean mutagenesis included carnation (*Dianthus caryophyllus*), Chrysanthemum (*Dendranthema Grandiflora*), and plants of Verbena sp. Afterward, several color and shape variations of petunia, Dahlia, and Torenia were also developed using this mutagenesis technique. Furthermore, the varieties developed using Ion beam mutagenesis include not only ornamental plants of high commercial demand [46], but also crops like salt-tolerant rice, citrus fruits, coniferous trees, mutant blast-resistant rice [47], mutant muskmelon, and rice varieties with lower fertilizer requirements [48].

#### **3.3 Chemical mutagenesis**

Chemical mutagenesis is the most efficient and expedient tool used for a large number of plant species. Ethyl methane sulfonate and sodium azide are the most widely used chemical mutagens to induce mutations in various crop plants like a tomato. The chemical mutagens used in mutation breeding are ethyl methanesulphonate (EMS), hydroxylamine, methyl methanesulphonate (MMS), sodium azide hydrogen fluoride (HF), and N-methyl-N-nitrosourea (MNU) [32]. Although, EMS is the most extensively used mutagen in plants due to its high efficiency at inducing point mutation (changes in a single nucleotide) and deletions (loss of chromosomal segment) in the chromosomal fragments. Mutant populations in various cereal crops using chemical mutagens for seeds or pollens have been developed comprising maize [49], barley [50, 51], rice [52], sorghum [53], and both hexaploid bread wheat [54] and durum wheat [55]. The EMS was exploited for potyvirus resistance in tomato [23].
