Keywords

1 Introduction

Globally , the current human population is increasing day by day and expected to reach 9 billion by 2050 and that will lead to food scarcity on earth since 70 % more food will be required to meet this challenge. To overcome this increasing demand for food and proper nourishment, an improvement in food production is urgently needed (Ronald 2014). According to the Food and Agriculture Organization (FAO) of the United Nations, food security exists when all people, at all times, have physical, social and economic access to sufficient safe and nutritious food that meets their dietary needs and food preferences for an active and good life (FAO 2014a).

The envisaged increase in food production is daunting because of limited available arable land, depleting water resource and varying climatic conditions. The difficulties are also compounded by urbanization, salinization, biotic stress , drought and desertification that result in a reduction of arable land. Moreover, changing climatic conditions and subsequent variations also limit food production (UNEP 2002). Agrochemicals cannot be used excessively to meet the challenge of food shortage due to their deleterious impacts on health and environment (Mba 2013). FAO (2011) recommended high-yielding varieties to meet the food shortages, as well as efficient use of input, under the projected climate change and adapted to a wide range of agroecosystems conditions. Food production must be increased through fewer agricultural inputs with maximum environmentally-friendly methods (Schlenker and Roberts 2009). Keeping in view the limited arable land, water resources and variable climatic conditions, new crop varieties and cropping system that use more efficiently the limited resources have to be developed to meet the challenges of food security.

There are different mechanisms for harnessing the heritable variations encoded in the genetic make-up of existing crop plants so as to use them in crop improvement programs. The incorporation of desired traits from non-adapted landraces /crop wild resource can speed up crop improvement. Putative parental material can also be induced to mutate so as to obtain new genes that control desired traits for new crop variety development (Suprasanna et al. 2014).

Among the different strategies to enhance crop improvement programs, induced mutagenesis has contributed immensely by creating mutant varieties with improved and desirable genetic changes in agronomically-important traits of the crop plants. Mutagenesis has become more efficient in combination with advanced molecular biology techniques and in vitro culture methods that result in enhancement of crop improvement/breeding programs (Jain 2010a) particularly under the global climate change (Jain 2010b). Such induced mutagenesis also helps in the mining of new gene alleles that do not occur in the germplasm (Roychowdhury and Tah 2013).

2 Induced Mutagenesis

Mutation is a term coined by De Varies (1901) upon the appearance of a new phenotype he noted in the common evening primrose, Oenothera lamarkiana, to describe the sudden heritable change in the genotype of an organism; the organisms with such heritable changes are known as mutants (Mba 2013). Mutation is the ultimate source of all genetic changes which provide the raw material for evolution and it is a valuable approach for improvement of economic characters of plants. Such genetic changes can occur spontaneously naturally at a very low rate or experimentally induced by physical and chemical mutagens (Jain 2002, 2010a; Mba et al. 2007). Physical mutagens include radiations such as α-rays, β-rays, fast neutrons, thermal neutrons, x-rays , γ-rays and ultraviolet (UV) radiation. Most common chemical mutagens include alkylating agents, such as ethyl methane sulphonate (EMS ); methyl methane sulphonate (MMS); ethylene imines (EI); diethyl sulphate (DES) etc. Also, azides e.g. sodium azide ; acridine dyes e.g. acriflavine, acridine orange, proflavin etc.; base analogues e.g. 5 bromouracil, 2 aminopurine, 5 chlorouracil, etc.; and other direct-acting chemicals such as nitrous acid, mustard gas etc.

3 Spontaneous Mutation

Spontaneous mutations in crop plants occurs naturally during adaptations and evolutionary processes at an extremely low rate i.e. 10−5–10−8 . This frequency is inadequate for creating variations in the genetic architecture of a crop for improvement of desirable traits (Zhong-hua et al. 2014). Wheat , peas and barley are the notable example of mutants derived through heritable permanent change i.e. spontaneous mutations during the course of domestication (Table 4.1). Spontaneous mutations in these plants resulted in eradicated pod or head shattering and the reductions in seed dormancy periods. Other examples of spontaneous mutants include those found in almond, lima bean, watermelon, potato, eggplant, cabbage and several types of nuts (Mba 2013).

Table 4.1 Spontaneous mutations and domestication of crops
Full size table

High yielding and lodging resistance in wheat varieties were developed by the incorporation of spontaneously-mutated alleles of the genes that resulted in the Green Revolution and subsequently secured food for millions of people around the world. Other examples include utilization of dwarf germplasm Dee-geo-woo-gen from China and the release of rice variety IR8 developed in the Philippines by the International Rice Research Institute (IRRI) from a dwarf line (Mba et al. 2012b). Application of cytoplasmic male sterile (CMS) and photoperiod-sensitive genic male sterile rice lines were utilized to develop hybrid rice seeds for commercial release (Mba and Shu 2012). Therefore, induction of mutations in existing crop plant germplasm will be helpful to create maximum genetic variability, to identify new genes/alleles of desired interest and lead to a scaling-up of crop improvement programs.

4 Induced Mutation

Over the last six decades, thousands of new mutant crop varieties with improved agronomic characters have been developed by induced mutation s throughout the world (Jain and Maluszynski 2004). After the epoch-making discoveries made by Muller (1927) and Stadler (1928a, b), a large amount of genetic variability has been induced by various mutagens /radiation in different crop plant species that have resulted in the development of more than 3218 officially-released mutant varieties (Figs. 4.1 and 4.2) worldwide in about 224 plant species during the past 60 years (FAO 2014b). Among the mutant varieties the majority are of food crops contributing to environmentally-sustainable food security in the world. In India 343 mutant varieties, by using different mutagens , have been released for cultivation (Tables 4.2 and 4.3).

Fig. 4.1
figure 1

Registered mutant crop varieties (FAO 2014b)

Full size image
Fig. 4.2
figure 2

Distributions of mutant crop varieties by continents of official release (Source: FAO 2014b)

Full size image
Table 4.2 Mutant varieties of different crops released for cultivation in India
Full size table
Table 4.3 Mutagens used and trait improved in mutant cultivars released in India
Full size table

For enhanced food production , there is an urgent need to create genetic variability in the desired crop plant species. This genetic variability could be used in the development of new varieties with increased yield, disease and lodging resistant, modified protein, oil and starch content, increased nutrient uptake, deeper rooting system and resistant to abiotic stresses such a drought, heat and salinity etc. to counter the erratic climate conditions that limit crop production (Jain 2002, 2010b).

4.1 Physical Mutagenesis

After the pioneering work using X-rays , by Muller (1927) and Stadler (1928a, b) on fruit fly and maize , scientist began to create genetic variations by using radiation and it became a very prominent method (Ahloowalia et al. 2004). Among physical mutagens , ionizing radiations i.e. gamma rays and X-rays are the most commonly used methods (Mba and Shu 2012; Mba et al. 2012a). Other physical mutagens include alpha (α) and beta (β) particles and fast neutrons to induce genetic changes in crop plants (Table 4.4). These mutagens cause deletion or addition of nucleotides and by substitution of one or more nucleotides for the new combinations of genes (Mba 2013).

Table 4.4 Different physical mutagens and their properties
Full size table

With pioneering work in China and Japan (Mei et al. 1994a; Wu et al. 2001), an ion beam , generated by particle accelerators, i.e. cyclotron using 20Ne, 14N, 12C, 7Li, 40Ar, or 56Fe as radioisotope sources, has gained popularity in the induction of mutations. Cosmic radiations have also been used to induce mutations in crop plants (Liu et al. 2007; Mei et al. 1998; Ren et al. 2010). Other researcher found fast neutron bombardment most efficient in mutation induction (Koornneef et al. 1982). Among these, gamma rays are less destructive and result in large deletions (Yuan et al. 2014) or small deletions while translocations, chromosome losses and large deletions have been induced after fast neutron bombardment (Sikora et al. 2011). Li et al. (2001) generated 51,840 lines of Arabidopsis population by fast neutron mutagenesis used for screening of mutants, while in rice around 10,000 rice mutant lines have been generated by fast neutron bombardment and around 20,000 lines by γ-ray. (Wu et al. 2005).

4.2 Chemical Mutagenesis

Chemical mutagenesis is the use of chemical compounds that can induce mutations (Table 4.5). The demonstration that nitrogen mustard gas caused mutations in the cell (Auerbach 1940, 1947; Auerbach and Robson 1944, 1946) opened the way for identification of chemicals which induce mutations. Plant breeders and geneticists applied chemical mutagens to induce mutations in various crop plants because they are easy to use and no special equipment is required to induce high mutation frequency (Gulfishan et al. 2012, 2013; Henikoff and Comai 2003; Koornneef et al. 1982; Zhu et al. 1995). The chemical mutagen reacts with DNA of the treated seed/cell/tissue or organ and induces somatic and genetic changes and only unrepaired damage to the DNA in initial cells of the sporogenic layer (germ line cells) are transferred as mutations to the next generation. Other mutations in somatic cells of the embryo, including mitotic chromosomal aberrations together with toxic action of mutagen on all components of cytosol, affect plant growth and development, and are called the somatic effect of the mutagen (Roychowdhury and Tah 2013). Compared to radiation, chemical mutagens tend to cause single base-pair (bp) changes, or single-nucleotide polymorphisms (SNPs) rather than deletions and translocations of nucleotide (Sikora et al. 2011). Chemicals include base analogues, alkylating agents, azides and others that modify genetic makeup of crop plants. Among chemical mutagens ethyl methane sulphonate (EMS ) has been used frequently. It causes point mutations such as nonsense, missense and silent mutations by chemical modification of nucleotides within the DNA (Jiang and Ramachandran 2010). EMS mainly induces C to T changes that results in substitution of C/G to T/A (Kim et al. 2006; Krieg 1963) and at lower doses it induces G/C to A/T transversions through 7-ethylguanine hydrolysis or A/T to G/C transitions through 3-ethyladenine pairing errors (Greene et al. 2003).

Table 4.5 Frequently used chemical mutagens and their properties
Full size table

5 Other Methods for Induction of Mutation

In recent years, heavy ion irradiation has also attracting attention as an effective method of induction of mutation in plants. These heavy ion beams possess high linear energy transfer (LTE) and relative biological effectiveness (RBE) which is supposed to enhance mutation frequency and also induces wide spectrum of mutations at low doses and short duration of treatments (Abe et al. 2012; Tanaka et al. 2010). Hirano et al. (2012) in Arabidopsis and Mei et al. (1994b) in rice investigated the effectiveness of ion beams and concluded that they were more effective as compared to gamma rays and X-rays . In Japan, mutations have been induced by using heavy ion beam irradiations (Hase et al. 2010; Kondo et al. 2009; Tanaka et al. 2010) and in China low energy ion beam have also been used to create mutations (Feng et al. 2009). Bacterial leaf blight and blast resistance in rice (Xiao et al. 2008), yellow mosaic virus in barley (Tanaka et al. 2010) and potato virus Y in tobacco (Hamada et al. 1999) have also been induced by using ion beam radiations. Resistance to sigatoka disease in banana has been induced by carbon ion-beam irradiation (Reyes-Borja et al. 2007). The UVB resistant rice mutant, utr319, was isolated after carbon ion irradiation which was more tolerant to UV than the wild type (Takano et al. 2013). Chundet et al. (2012) created rice mutant expressing short stature and photoperiod insensitivity by using a low-energy N+/N2 + ion beam (Fig. 4.3). A chlorophyll deficient mutant was also isolated in Arabidopsis thaliana by carbon ion irradiation (Shikazono et al. 2003). Electron beams induced mutation s of leaf shape and color, seed size and shape, trailing, more branching, dwarfing, early or late flowering time and high yield in Vigna angularisi (Luo et al. 2012). Tanaka et al. (2010) reviewed the work on ion beam irradiation on different plant and found that the spectrum of mutations induced by ion beams was different than gamma rays . In rice, the mutation spectrum of different ion beams was similar to that of gamma rays but the frequency of mutations was higher (Yamaguchi et al. 2009). Luo et al. (2012) isolated leaf shape mutants in Vigna angularisi by electron beam irradiations (Fig. 4.4). In Chrysanthemum ray floret mutants has been induced by 12C5+ ion beam irradiation as shown in Fig. 4.5 (Matsumura et al. 2010).

Fig. 4.3
figure 3

Morphological mutants in KDML105 jasmine rice induced with a low-energy N+/N2 + ion-beam. (a) and (b) illustrate the wild-type (WT) plant versus the mutant (BKOS) phenotype . The mutant has a purple color in the plant stem (c), Leaf (d), immature-seeds (e), husked (f) and dehusked mature seeds (g) (Source: Chundet et al. 2012)

Full size image
Fig. 4.4
figure 4

Mutants of leaf shape induced by electron beam. (a) Jingnong 6 control. (b) Sword leaf (600 Gy from Jingnong 6). (c) Lanceolate leaf (600 Gy from Jingnong 6) (Source: Luo et al. 2012)

Full size image
Fig. 4.5
figure 5

Ray floret mutants of Shiroyamate obtained by 12C5+ ion beam irradiation . (a) Original flower of Shiroyamate; (b) yellow mutant; (c) pale-yellow mutant (Source: Matsumura et al. 2010)

Full size image

In recent years, cosmic radiations have also been recognized as a source of induction of mutations in plants and the term space breeding has been coined for such breeding programs. Mutations were induced in seeds on space flights (Kranz 1986; Mei et al. 1994b, 1998). Scientist used space craft, satellites and high altitude balloons to carry seeds into space for mutation induction (Li et al.1999), and in the subsequent generation of plants genetic changes have been recorded (Guo et al. 2010; Hu et al. 2010; Ou et al. 2010). Space flight cauliflower experienced changes in plant size and flower head and also resistance to black rot (Wua et al. 2010). Blast-resistant rice mutants have also been developed by using space flight mutagenesis (Xiao et al. 2008). Since 1987, China has developed more than 40 mutant varieties in different crop plant such as rice, wheat , cotton, pepper and tomato by space mutagenesis (Chengzhi 2011).

Plant cell and tissue culture have helped plant breeders in creating genetic variation in plants. Efficiency of mutation induction has been improved by the development of in vitro techniques (Ahloowalia 1998; Jain 2001, 2006a, 2007). Easy handling of the regenerated population and the wide range of plant material for mutagenic treatment are the main advantage of in vitro mutagenesis (Jain 2000; Predieri 2001) In vitro culture is useful in creating variation specially in vegetatively propagated plants such banana (Chai et al. 2004; Roux et al. 2009) cassava (Jain 2005) sweet potato (Ahloowalia 1997; Ahloowalia and Maluszynski 2001) sugarcane (Kenganal et al. 2008; Patade et al. 2008; Suprasanna et al. 2009) citrus species, (Predieri 2001; Somsri et al. 2008) indiangrass (Stephens 2009) and ornamental species (Ahloowalia 1997; Jain et al. 2006a, b). Jain (2010a) isolated different types of mutants in banana such as large fruit size, reduced height, early flowering and resistant to Fusarium wilt and black sigatoka by in vitro mutagenesis. Bayoud disease resistant mutants were developed in date palm by in vitro mutagenesis (Jain 2012).

6 Potential Use of Mutants in Crop Improvement

6.1 Mutants as a Raw Material for Crop Improvement

Mutants have long been used by scientists directly or indirectly (cross breeding) for the development of new varieties. Efforts have been made by plant scientists to set up experimental protocols to create heritable genetic variations among crop plants and their subsequent use in crop improvement programs. A mutant varieties database (MVD) is maintained by FAO/IAEA (http://mvgs.iaea.org/AboutMutantVarities.aspx), containing newly-introduced rice varieties of modified zinc and starch content and reduced grain size from China, and an early-maturing, flood- and disease-resistance variety of rice, BINA DHAN-7, from Bangladesh. Most of the mutant varieties (77 %) are seed propagated and approximately (48 %) varieties recorded in the MVD are cereals , with rice at the highest constituting 53 %, followed by barley at 20 %. Induced mutants are freely available for crop improvement programs without restrictions on their use in contrast to protected plant varieties or germplasm. Many mutants have been released directly as new varieties and many others used as parents to create varieties with improved traits such as improved yield, quality of seed propagated crops, modified oil, protein and starch quality, enhanced uptake of specific metals, deeper rooting system and resistance to biotic and abiotic stresses . Outstanding mutant varieties such as rice in Australia, India, China, Pakistan and Thailand; cotton and wheat in Pakistan; pear in Japan; grapefruit, sunflower and peppermint in the USA; barley in several countries of Europe; durum wheat in Italy; sorghum in Mali; groundnut and pulses and several ornamental plants in India, the Netherlands and Germany have been grown successfully and are playing pivotal roles to keep food scarcity at bay (Ahloowalia et al. 2004). Mutant germplasm stock for different plants has been maintained by several countries round the world (Table 4.6) for their use in future crop improvement. The contribution of mutant crop varieties, cultivated worldwide, in food security were reviewed by Kharkwal and Shu (2009) and they concluded that mutant varieties contribute significantly to ensuring food and nutritional security by enhanced resistance to biotic and abiotic factors, higher yield, improved nutrient use efficiencies and less agricultural inputs. All these qualities have made mutant varieties an integral part of daily diet around the world. Promising characteristics of the mutant varieties have been shown in Fig. 4.6.

Table 4.6 Mutants/germplasm stocks of crops and the host institutions
Full size table
Fig. 4.6
figure 6

Characteristics of mutant varieties

Full size image

6.2 Resource for Genomics and Molecular Breeding

Induced mutagenesis coupled with molecular biology techniques have made it possible to generate large mutant populations in different crops (Caldwell et al. 2004; Krishnan et al. 2009; Wang et al. 2013; Xin et al. 2008). DNA sequencing , genome wide association analysis, transcriptomics and proteomics data of such mutant populations have been used for functional genomics studies. These mutant populations have also been used for gene discovery by forward and reverse genetic approaches. In rice , 64 genes have been discovered so far that are responsible for mutant phenotypes in plant development, photosynthesis, signaling transduction and disease resistance (Morrell et al. 2012). Knowledge of functions, expression and regulations of these genes responsible for agronomically-important phenotypes will benefit crop improvement .

Induced mutants play pivotal roles in plant breeding . Mutation breeding , by using EMS , fast neutron and gamma irradiation , created 443 rice cultivars (Kharkwal and Shu 2009). In 1976, Calrose 76, the first semi-dwarf rice cultivar, was developed in the USA and afterward numerous semi-dwarf cultivars and other useful mutants with improved characters, such as early maturity, endosperm quality, elongated uppermost internode and genetic male sterile, improved nutritional quality because of low phytic acid, giant embryo mutants of potential interest to the rice oil industry and adapted basmati and jasmine germplasm were developed by using Calrose 76 (Rutger 2009). In maize , a model biological system and also an important agronomic crop, shrunken2 (sh2) mutant kernels were sweet (Laughnan 1953). By using this mutant as a parent in maize improvement programs sh2-based sweet varieties have been developed (Tracy 1997). Amylose extender1, Leafy1, Sugary1, sugary enhancer1, waxy1, opaque2, floury2, and brown midrib3 are other maize mutants that have also been used directly for specialty maize production (Cox and Cherney 2001; Hallauer 2001). Mutant germplasm collections, for a specific character, have also been maintained such as in barley ; more than 700 anthocyanin and proanthocyanidin mutants were used for identifying genes involved in the flavonoid metabolic pathway in barley (Jende-Strid 1993). One more barley mutant cultivar, Diamant, has been used for breeding more than 150 leading barley cultivars all over the world (Ahloowalia et al. 2004). High oleic acid mutants of sunflower were widely used in the USA and Europe, low linolenic acid and high oleic acid mutants of rapeseed induced in Canada have been used for breeding rapeseed in Australia, Canada and Europe (Ahloowalia et al. 2004).

7 Impact of Mutant Varieties

Over the past six decades, more than 3000 mutant varieties have been developed by mutation induction in more than 200 plant species. Rice , wheat , cotton, rapeseed, sunflower, sesame, grapefruit and banana are economically-important crops with a large number of mutant varieties. Among these varieties, rice in Australia, China and Thailand; rice and cotton in Pakistan; Japanese pear in Japan; barley varieties in Europe; durum wheat in Italy; sunflower, grapefruit and peppermint in USA; sorghum in Mali; rice, groundnut, pulse crops and ornamentals in India, the Netherlands and Germany, have all had a positive impact on the economy of the individual countries (Ahloowalia et al. 2004). Rice varieties resistant to salinity, and with early-maturing and high-quality traits, have been developed in Vietnam providing extra income to the farmers and generating USD 300 million per year (Jain and Suprasanna 2011). Biotic and abiotic factors hinder crop production and these can be overcome by changing the crop genetic architecture that neutralizes the effect of biotic and abiotic stresses . Reduced height in cereals (Kharkwal and Shu 2009); bushy mutant in chickpea (Gaur et al. 2008) cotton (Ahloowalia et al. 2004) and sunflower (Jambhulkar and Shitre 2009) etc. are examples of changed genetic architecture of these plants. A flax mutant with low linola content and a sunflower mutant with high oleic acid, developed by induced mutagenesis , improve the quality of the product. NIAB Karishma a cotton leaf curl virus resistant cultivar, generates USD 294.4 million in Pakistan (Haq 2009). Calrose 76, a semi-dwarf rice mutant developed through gamma irradiation , has 14 % more yield than the wild type and an added USD 20 million per year to farmers (Rutger 2006). Golden Promise and Diamant barley mutants brought millions of dollars to the malting and brewing industry in Europe.

8 Conclusions and Prospects

Currently available staple crops are insufficient to meet the challenges of the twenty-first century and provide total world food security. Enhanced food production , together with reduced postharvest loses are primary approaches to feed the expected 9 billion population by 2050. Induced mutagenesis has played an important role by creating several mutants in different crop plants. These mutant varieties with specific character/trait such as high yield, resistance to biotic and abiotic stresses , have been grown globally bringing a significant positive economic impact and contribute to global food and nutritional security and improved livelihoods. Despite the available mutant resources , challenges still lie ahead to feed an ever-increasing population. To speed up crop production, mutant resources for different crop plants have to be established which can be used to create new mutant cultivars which are high yielding, resistant to biotic and abiotic stresses , enhanced uptake of specific metal, deeper rooting systems and modified oil, starch and protein content that can boost industrial processing. Now in the nanotechnology era, scientists develop can new tools and techniques for crop improvement . Recently, nanoparticles, nanocapsules and nanofibers have been utilized in gene manipulation for crop productivity enhancement. Numbers of biological and chemicals materials applied as a functionalized nanoform to regulate gene expression in plant systems. Nanofiber-based delivery of genetic materials is the best alternative process of a microinjection gene delivery method. Carbon nanofiber arrays are being used for fast and efficient delivery of genetic material in plant cells in crop engineering. With the innovation of molecular biological and nano-level techniques, it is now possible to study more agronomically-important traits at the molecular level that will help in creating envisaged smart crop varieties that overcome the constraints threatening twenty-first century global food security.