**2. Application of gamma rays to induce mutation and** *in vitro* **selection**

As mutations may occur spontaneously, it can induce artificially. Artificially induced mutations can be created by physical mutagens, such as X-rays, gamma rays, and neutrons, and chemical mutagens, such as ethyl methanesulfonate (EMS), in plant mutation breeding [12]. Although, various mutagens (physical and chemical) are used for the induction of mutation, physical mutagens (radiation: gamma rays and X-rays) was used more frequently as compared to chemical mutagens. However, among the physical mutagens, gamma rays (1604 improved mutants) are used more widely than X-rays (561 improved mutants). Gamma rays are ionizing radiation and used in inducing mutations in seeds and other planting materials such as cuttings, pollen, or tissue-cultured calli [4, 13]. In the early twentieth century, plant biologists discovered method that the frequency of genetic variations could increase in treated seeds with chemical compounds or radioactive rays. Mutation induction has become a powerful tool for developing new and novel plant germplasm [14]. Radiation-induced mutation is one the most widely used method to improve direct mutant varieties compared to acclimatization, selection, hybridization, which are laborious, time consuming, and also with limited genetic variation [15]. This discovery later referred as plant mutation breeding. As an *in vitro* culture techniques, *in vitro* selection can be use to obtain plant genotype tolerance to adverse environment conditions such as drought, high salinity [16]. Numerous mutant or genetic material is possible due to *in vitro* selection [17]. *In vitro* selection is the greatest method to obtain the desired traits of plant, because it has the capacity to manipulate the variation to the expected result. Both abiotic and biotic tolerant plants could be obtained from the media which contain the selection agent [16]. *In vitro* selection has been practiced for desirable traits and success has been achieved in several crop plants [18–20]. Easy application of *in vitro* selection method is one of the most important requirements for the achievement of *in vitro* selection technique and to obtain the tolerant plants. Exposed biological materials by mutagens can be reliable and easy screening in a comparatively small space, which can save time, money, and space under *in vitro* conditions when comparing the greenhouse and field [21].

researchers reported spontaneous variation in plants between 1590 and 1968 [6]. However, the first publications of induced mutations (through X-rays) for the plant breeding was published 89 years ago by Muller and Stadler [7, 8]. As a result of these studies, mutation breeding as a new approach was added to other plant breeding methods. Thereafter, a large amount of genetic variability has been induced by various mutagens (physical and chemical) and contributed to modern plant breeding. *Nicotiana tabacum* was the first commercial mutant cultivar (called: chlorine type), which produced by inducing mutations [6]. Induced mutations were used to improve tolerant plant varieties over the past 50 years in all over the world [8]. Currently, 3246

Increasing crop yields is a major demand for assuring food security. Mutagenesis is an important tool to improve crops and has not got any regulatory restrictions as genetically modified organisms (GMOs) [10]. Plant breeding is based on the genes. Initially, breeders select new phenotypes with valuable characters without knowing the genetic constitution. The emergence of molecular genetics is parallel to understand the details of inheritance of desirable/undesirable traits and genetically controlled, modern biotechnological breeding has paved a wide road. By using DNA recombinant technologies, the gene encoding a trait precisely manipulated to create novel phenotypes. The cloned gene in respect of the source or recipient of the genes can be transferred by breeding technology known as transgenic technology. In transgenic technology, the key step is the integration of desired foreign genes into the host plant genome. For plant transformation, there are primary tree methods such as the Agrobacterium-mediated, particle bombardment, and protoplast transformation. The Agrobacterium-mediated gene transfer method is one of the most practical and suitable method [2]. The first transgenic plant (tobacco [*N. tabacum*], which contain antibiotic resistance gene) was obtained in 1980 by Marc De Block through Agrobacteriummediated method [11]. Breeders can transfer encoding genes of new characters into plants genome through transgenic approaches. Its precision and the betterment of a trait without changing the genetic makeup of genome in elite genotypes are the main advantages [2]. Although transgenic technology has significant achievement in improving crops and has substantial commercial value, this technology has some technical obstacles. For example, in terms of highly recalcitrant to genetic transformation and regeneration, there are many economically important plant species or elite varieties of species. In addition to the technical obstacles, there are some debates about unpredictable risks of transgenic technology on environment and food safety, even though many of these debates are baseless. However, more advanced technologies have been developed to solve these ideas [2]. On the other hand, plant breeders use mutagenesis in plant breeding programs without restrictions such as the legislative constraints, licensing costs, and societal opposition of transgenic technology [10]. Although still limited to the content of the endogenous genome, mutagenesis and high-resolution screening will supply a very good complement to recombinant DNA technologies and genetically modified organisms (GMOs) in further improved new plant forms that are better

registered mutagenic plant varieties are there in FAO/IAEA mutant data base [9].

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adapted to change conditions of environment and the increasing global population [1].

**2. Application of gamma rays to induce mutation and** *in vitro* **selection**

As mutations may occur spontaneously, it can induce artificially. Artificially induced mutations can be created by physical mutagens, such as X-rays, gamma rays, and neutrons, and chemical Beyaz [22] reported easy and reliable *in vitro* selection protocol and optimization for the creation of irradiation-based mutagenesis in sainfoin (*Onobrychis viciifolia* Scop.). Sainfoin (*O. viciifolia*) is a significant forage legume species, which grown widespread than the other *Onobrychis* species, agriculturally [23]. The seeds of sainfoin (*O*. *viciifolia* Scop.) were irradiated to 0, 400, 500, and 600 Gy using an experimental 60Co source (dose rate of 0.483 kGy h−1) at the Sarayköy Nuclear Research and Training Center (SANAEM) of the Turkish Atomic Energy Authority (Ankara, Turkey). Irradiated and unirradiated (control) seeds of sainfoin were sowned into Murashige and Skoog (MS) medium supplemented with 150 mM NaCl (**Figure 1A**). Seeds were allowed to germinate and develop at 20 ± 1°C under cool white fluorescent light (27 μmol m−2 s−1) with a (16 h light/8 h dark) illumination period for 10 days. After that, selected plantlets were transferred to MS medium without NaCl for continuous development for 2 weeks (**Figure 1B**). For acclimatization, following steps were applied. Advanced plantlets (**Figure 1C**) were transferred to plastic glasses (**Figure 1D**). After that, plastic glasses placed in freezer bags (**Figure 1E** and **F**) to provide suitable moisture and adaptation to external condition for 1 week grown in the plant growth chamber. Plantlets in freezer bags were transferred to plant growth chamber (**Figure 2A**) and the mouths of the freezer bags slowly open during 1 week period, and at the end of 1 week, all plantlets were removed from freezer bags (**Figure 2B**). Every 2 days for 2 weeks, acclimatized plantlets watered with 30 ml tap water containing 150 mM NaCl for *in vivo* selection. Most of the plantlets died (**Figure 2C**) during treatments of NaCl, survived plantlets were selected (**Figure 2D**). Survived plantlets (**Figure 2E**) were transferred to plastic glasses which contain soil without NaCl for more

**Figure 1.** Irradiated and unirradiated seeds of sainfoin in MS-basal selection media supplemented with 150 mM NaCl (A). After 10 days of seed germination, selected plantlets and advanced plantlets in MS-basal medium without NaCl (B, C). An acclimatization process of plantlets to external conditions (D–F).

**A B** 

36 Plant Engineering

**C D** 

**E F** 

C). An acclimatization process of plantlets to external conditions (D–F).

**Figure 1.** Irradiated and unirradiated seeds of sainfoin in MS-basal selection media supplemented with 150 mM NaCl (A). After 10 days of seed germination, selected plantlets and advanced plantlets in MS-basal medium without NaCl (B, **Figure 2.** General view of plantlets in freezer bags (A). Plantlets which removed freezer bags in plant growth chamber (B). Dead plants as a result of salt application (C). Survived plantlets (D). Survived plantlets after treatments of NaCl (E). Washing root of survived plants with tap water to get rid of NaCl and transferred to new soil without NaCl (F and G).

development (**Figure 2E** and **G**). Superior plants (putative mutants) in terms of salt tolerance were moved to a growth chamber (**Figure 3A** and **B**) for a while, and after that, they were transplanted in field (**Figure 3C**–**F**).

**Figure 3.** General view of survived plants of sainfoin in growth chamber (A and B). Transferred putative mutants against to salt stress in the field (C and D). Putative mutant sainfoin plants in the field after 1.5 months (E) and 2 months (F) (Location: The University of Ankara, Faculty of Agriculture, Department of Field Crops, Turkey, photos were taken on 21 March 2013).
